pUNO-mRIG-I
A plasmid expressing the murine RIG-I (DDX58) gene
Catalog # puno-mrigi
For research use only
Version # 05G22-SV
PRODUCT INFORMATION
Content:
- 1 disk of lyophilized GT110 E. coli bacteria transformed with pUNO-mRIG-I.
- GT110 genotype is: F-, mcrA , ∆(mrr-hsdRMS-mcrBC) , Ø80lacZ∆M15 ,
∆lacX74, recA1 , endA1.
- 4 pouches of E. coli Fast-Media® Blas.
Storage and stability:
- Products are shipped at room temperature.
- Transformed bacteria should be stored at -20°C and are stable up to 1 year.
- Store E. coli Fast-Media® Blas at room temperature. Fast-Media® pouches are
stable 18 months when stored properly.
Quality control:
- Plasmid construct has been confirmed by restriction analysis and sequencing.
- Bacteria have been lyophilized, and their viability upon resuspension has
been verified.
GENERAL PRODUCT USE
pUNO are ready-made expression vectors containing a Toll-Like Receptor
(TLR) gene, a TLR-associated gene (co-receptor, adaptor, involved in TLR
signaling, etc.), or gene otherwise implicated in the innate immune response.
TLRs play a critical role in early innate immunity to invading pathogens by
sensing microorganisms. These evolutionarily conserved receptors recognize
highly conserved structural motifs only expressed by microbial pathogens,
called pathogen-associated microbial patterns (PAMPs). PAMPs include
various bacterial cell wall components such as lipopolysaccharides (LPS),
peptidoglycans and lipopeptides, as well as flagellin, bacterial DNA and viral
double-stranded RNA. Stimulation of TLRs by PAMPs initiates a signaling
cascade that involves a number of proteins, such as MyD88 and IRAK. This
signaling cascade leads to the activation of the transcription factor NF-κB
which induces the secretion of pro-inflammatory cytokines and effector
cytokines that direct the adaptive immune response.
pUNO may be used for:
Obtaining a TLR-related gene to subclone into another vector. Two
restriction sites flank the gene, allowing convenient excision. These restriction
sites are compatible with many restriction sites contained in multiple cloning
sites, thus facilitating subcloning.
Stable gene expression in mammalian cells. pUNO plasmids can be used
directly in traansfection experiments both in vitro and in vivo. pUNO plasmids
contain the blasticidin resistance gene (bsr) driven by the SSV40 promoter in
tandem with the bacterial EM7 promoter. This allows the amplification of the
plasmid in E. coli AND the selection of stable clones in mammalian cells. pUNO
allows a high level of expression and secretion of the gene product.
The mRIG-I (DDX58) gene may be cut out using Age Ior BspH I and Xba I
enzymes.
Age I is compatible with Xma I, BspE I, NgoM IV and SgrA I.
BspH I is compatible with Nco I and BspLU11 I.
Xba I is compatible with Nhe I, Spe I, and Avr II.
PLASMID FEATURES
• hEF1 / HTLV prom is a composite promoter comprising the Elongation Factor1α (EF-1α) core promoter1 and the R segment and part of the U5 sequence (RU5’) of the Human T-Cell Leukemia Virus (HTLV) Type 1 Long Terminal
Repeat2. EF-1α is an ‘housekeeping’ gene ubiquitously expressed in eukaryotic
cells. The EF-1α promoter exhibits a strong activity, higher than viral promoters
and, on the contrary to the CMV promoter, yields persistent expression of the
transgene in vivo. The R-U5’ has been coupled to the EF-1α core promoter to
enhance stability of DNA and RNA.
TECHNICAL SUPPORT
Toll free (US): 888-457-5873
Outside US: (+1) 858-457-5873
E-mail: info@invivogen.com
Website: www.invivogen.com
mRIG-I (DDX58) gene:
Intronless ORF from the ATG to the stop codon.
ORF size: 2781 bp.
Cloning fragment size: 2803 bp.
• SV40 pAn: The Simian Virus 40 late polyadenylation signal enables efficient
cleavage and polyadenylation reactions resulting in high levels of steady-state
mRNA.
• Ori is a minimal E. coli origin of replication with the same activity as the
longer Ori.
• SV40 prom: The Simian Virus 40 promoter allows the expression of the
blasticidin resistance gene in mammalian cells.
• Bsr (blasticidin resistance gene): The bsr gene from B. cereus encodes a
deaminase that confers resistance to the antibiotic Blasticidin S. The bsr gene
is driven by the SV40 promoter in tandem with the bacterial EM7 promoter to
allow blasticidin selection in both mammalian cells and E. coli bacteria.
• bGh pAn: The bovine growth hormone (bGh) polyadenylation (pAn) signal
and a transcriptional pause are placed 3' of the blasticidin gene. The bGh pAn
has been shown to be as efficient as SV40 and HSV1tk polyadenylation signals
in many different cell types3. The use of bGH pAn minimizes interference and
possible recombination events with the SV40 polyadenylation signal. The
pause site prevents transcriptional interference and read-through.
References
1. Kim et al. (1990). Gene 2: 217-223.
2. Takebe et al. (1988). Mol. Cell Biol. 1: 466-472.
3. Goodwin et al. (1992). J. Biol. Chem. 23: 16330-16334.
METHODS
Growth of pUNO-transformed bacteria:
Use sterile conditions to do the following:
1- Resuspend the lyophilized E. coli by adding 1 ml of LB medium in the
tube containing the disk. Let sit for 5 minutes. Mix gently by inverting the
tube several times.
2- Streak bacteria taken from this suspension on an blasticidin LB agar plate
prepared with the E. coli Fast-Media® Blas agar provided (see below).
3- Place the plate in an incubator at 37˚C overnight.
4- Isolate a single colony and grow the bacteria in TB supplemented with
blasticidin using the Fast-Media® Blas liquid provided (see below).
5- Extract the pUNO plasmid DNA using the method of your choice.
Note: For long-term storage of the pUNO-transformed bacteria, prepare a
20% glycerol stock of the bacteria grown in the overnight liquid culture and
freeze at -80˚C.
Selection of bacteria with E. coli Fast-Media:
E. coli Fast-Media® Blas is a new, fast and convenient way to prepare
liquid and solid media for bacterial culture by using only a microwave.
Method:
1- Pour the contents of a pouch into a clean borosilicate glass bottle or flask.
2- Add 200 ml of distilled water to the flask.
3- Heat in a microwave on MEDIUM power setting (about 400Watts), until
bubbles start appearing (approximately 3 minutes). Do not heat a closed
container. Do not autoclave Fast-Media®.
4- Swirl gently to mix the preparation. Be careful, the bottle and media are
hot, use heatproof pads or gloves and care when handling.
5- Reheat the media for 30 seconds and gently swirl again. Repeat as
necessary to completely dissolve the powder into solution. But be
careful to avoid overboiling and volume loss.
6- Let agar medium cool to 45˚C before pouring plates. Let liquid media cool
to 37˚C before seeding bacteria.
Note: Do not reheat solidified Fast-Media® as the antibiotic will be permanently
destroyed by the procedure.
3950 Sorrento Valley Blvd. Suite A
San Diego, CA 92121 - USA
SgfI (11)
NotI (5562)
XhoI (5556)
AgeI (553)
EcoRI (5275)
BspHI (561)
XhoI (659)
XmnI (4854)
hEF1/HTLV prom
BspHI (4846)
AseI (4790)
EcoRV (928)
AseI (957)
Bsr
BbrPI (4779)
SmaI (4742)
AvrII (4719)
EM7
NcoI (4626)
mRIG-I (DDX58)
SV40 prom
SmaI (1243)
PacI (4384)
pUNO-mRIG-I
(5725 bp)
EcoRV (1591)
Ori
BspLU11I (3650)
NheI (2017)
PacI (3644)
SwaI (3634)
EcoRI (2072)
HpaI (3535)
XbaI (3364)
BglII (3117)
BamHI (2371)
150
SgfI (11)
1 GGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTA
101 GAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCC
201 GTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCC
301 GCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACC
401 GGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTT
BspHI (561)
AgeI (553)
501 TCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACCTGAGATCACCGGTCATCATGACCGCGGCGCAGCGGCAGAATCTGCAAGCATTCAG
1 Me t Th r A l aA l a G l nAr gG l nAs nLeuG l nA l a Ph eAr
XhoI (659)
601 AGACTATATCAAGAAGATTCTGGACCCCACCTACATCCTCAGCTACATGAGTTCCTGGCTCGAGGATGAGGAGGTGCAGTACATTCAGGCTGAGAAGAAC
1 3 gAs pT y r I l eLy s Ly s I l eLeuAs pP r oTh r T y r I l eLeuSe r T y r Me t Se r Se r T r pLeuG l uAs pG l uG l uVa l G l nT y r I l eG l nA l a G l uLysAs n
701 AACAAGGGCCCAATGGAAGCTGCCTCACTCTTCCTCCAGTACCTGTTGAAGCTGCAGTCAGAGGGCTGGTTCCAGGCCTTTTTGGATGCCCTGTACCATG
4 7 As nLy s G l y P r oMe t G l uA l aA l a Se r LeuPh eLeuG l nT y r LeuLeuLy s LeuG l nSe r G l uG l y T r pPh eG l nA l a Ph eLeuAs pA l a LeuT y r Hi sA
801 CAGGTTACTGTGGACTTTGTGAAGCCATCGAAAGTTGGGACTTTCAAAAAATTGAAAAGTTAGAGGAACACAGATTACTTTTAAGACGTTTAGAACCAGA
8 0 l a G l y T y r Cys G l yL euCys G l uA l a I l eG l uSe r T r pAs pPh eG l nLy s I l eG l uLy s LeuG l uG l uHi s A r gLeuLeuLeuAr gAr gLeuG l uP r oG l
EcoRV (928)
AseI (957)
901 ATTTAAGGCCACAGTTGATCCAAATGATATCCTTTCTGAACTATCCGAATGTTTGATTAATCAGGAATGTGAAGAAATCAGACAGATCCGAGACACTAAA
113 uPh eLysA l a Th r Va l As pP r oAs nAs p I l eLeuSe r G l uLeuSe r G l uCys Leu I l eAs nG l nG l uCys G l uG l u I l eAr gG l n I l eAr gAs pTh r Ly s
1001 GGGAGAATGGCAGGTGCGGAGAAGATGGCCGAATGTCTTATCAGATCCGACAAGGAAAACTGGCCAAAGGTCTTGCAACTTGCTTTGGAGAAAGACAACA
147 G l yA r gMe tA l a G l yA l a G l uLy s Me tA l a G l uCys Leu I l eAr gSe r A s pLy s G l uAs nT r pP r oLy s Va l LeuG l nLeuA l a LeuG l uLysAs pAs nS
1101 GCAAGTTTAGTGAATTGTGGATTGTTGATAAAGGTTTCAAAAGGGCTGAAAGCAAGGCTGATGAGGATGATGGAGCGGAGGCGTCCAGCATCCAGATTTT
180 er Ly s Ph eSe r G l uLeuT r p I l eVa l As pLy s G l yPh eLy s A r gA l a G l uSe r LysA l aAs pG l uAs pAs pG l yA l a G l uA l a Se r Se r I l eG l n I l ePh
SmaI (1243)
1201 CATTCAGGAAGAGCCAGAGTGTCAGAATCTCAGTCAGAATCCCGGGCCTCCTTCAGAAGCGTCTTCTAATAATTTACACAGCCCATTGAAACCAAGAAAT
213 e I l eG l nG l uG l uP r oG l uCys G l nAs nLeuSe r G l nAs nP r oG l y P r oP r oSe r G l uA l a Se r Se r A s nAs nLeuHi s Se r P r oLeuLy s P r oAr gAs n
1301 TACCAACTGGAGCTTGCCCTGCCTGCCAAGAAAGGGAAAAATACAATAATATGTGCCCCTACTGGTTGTGGAAAAACCTTTGTGTCGCTTCTTATATGTG
247 T y r G l nLeuG l uLeuA l a LeuP r oA l a Ly s Ly s G l yL ysAs nTh r I l e I l eCysA l a P r oTh r G l yCys G l yL y s Th r Ph eVa l Se r LeuLeu I l eCys G
1401 AACACCATCTTAAAAAATTCCCATGTGGACAAAAAGGGAAAGTGGTCTTCTTCGCTAACCAAATTCCTGTCTATGAGCAGCAGGCAACTGTGTTCTCACG
280 l uHi s Hi s LeuLy s Ly s Ph eP r oCys G l yG l nLy s G l yL y s Va l Va l Ph ePh eA l aAs nG l n I l eP r oVa l T y r G l uG l nG l nA l a Th r Va l Ph eSe r A r
EcoRV (1591)
1501 ATATTTTGAAAGACTTGGGTACAACATTGCGAGCATTTCTGGGGCAACATCTGATAGCGTCTCAGTGCAGCACATCATTGAAGACAATGATATCATCATC
313 gT y r Ph eG l uAr gLeuG l yTyrAs n I l eA l a Se r I l eSe r G l yA l a Th r Se r A s pSe r Va l Se r Va l G l nHi s I l e I l eG l uAs pAs nAs p I l e I l e I l e
1601 CTGACACCCCAGATTCTTGTGAACAATCTCAACAACGGAGCCATCCCCTCGTTGTCTGTCTTCACTCTGATGATATTTGATGAGTGTCATAACACTAGCA
347 LeuTh r P r oG l n I l eLeuVa l As nAs nLeuAs nAs nG l yA l a I l eP r oSe r LeuSe r Va l Ph eTh r LeuMe t I l ePh eAs pG l uCys Hi sAs nTh r Se r L
1701 AAAACCACCCATACAATCAGATCATGTTCAGATACCTAGACCACAAACTTGGAGAGTCACGGGACCCACTGCCTCAGGTCGTTGGGCTGACTGCCTCCGT
380 ysAs nHi s P r oTyrAs nG l n I l eMe t Ph eAr gT y r LeuAs pHi s Ly s LeuG l yG l uSe r A r gAs pP r oLeuP r oG l nVa l Va l G l yL euTh r A l a Se r Va
1801 CGGCGTTGGAGATGCTAAGACCGCGGAGGAAGCCATGCAACATATCTGTAAACTCTGTGCCGCCCTGGATGCCTCCGTGATTGCCACAGTCAGAGACAAC
413 l G l yVa l G l yAs pA l a Ly s Th r A l a G l uG l uA l a Me t G l nHi s I l eCys Ly s LeuCysA l aA l a LeuAs pA l a Se r Va l I l eA l a Th r Va l A r gAs pAs n
1901 GTTGCAGAACTGGAACAGGTCGTTTATAAGCCCCAGAAAATTTCCAGGAAAGTGGCATCCCGGACTTCGAACACGTTTAAATGCATCATCTCTCAGCTGA
447 Va l A l a G l uLeuG l uG l nVa l Va l T y r Ly s P r oG l nLy s I l eSe r A r gLy s Va l A l a Se r A r gTh r Se r A s nTh r Ph eLy s Cys I l e I l eSe r G l nLeuM
NheI (2017)
EcoRI (2072)
2001 TGAAGGAGACAGAGAAGCTAGCCAAGGATGTCTCCGAGGAACTTGGAAAGCTTTTTCAAATTCAAAACAGAGAATTCGGCACCCAGAAATATGAACAGTG
480 e t Ly s G l uTh r G l uLy s LeuA l a LysAs pVa l Se r G l uG l uLeuG l yL y s LeuPh eG l n I l eG l nAs nAr gG l uPh eG l yTh r G l nLy s T y r G l uG l nT r
2101 GATTGTCGGCGTCCACAAAGCGTGCTCAGTGTTTCAGATGGCAGACAAAGAGGAGGAGAGCCGGGTCTGCAAAGCGCTCTTCCTGTACACATCACATTTG
513 p I l eVa l G l yVa l Hi s LysA l a Cys Se r Va l Ph eG l nMe tA l aAs pLy s G l uG l uG l uSe r A r gVa l Cys LysA l a LeuPh eLeuT y r Th r Se r Hi s Leu
2201 CGGAAATACAACGATGCACTCATCATCAGTGAGGATGCACAGATGACAGACGCTCTAAATTACCTCAAAGCCTTCTTCCACGATGTCCGAGAAGCAGCAT
547 A r gLy s TyrAs nAs pA l a Leu I l e I l eSe r G l uAs pA l a G l nMe t Th r A s pA l a LeuAs nT y r LeuLysA l a Ph ePh eHi sAs pVa l A r gG l uA l aA l a P
BamHI (2371)
2301
580
2401
613
2501
647
2601
680
2701
713
2801
747
2901
780
3001
813
TCGATGAGACCGAGCGAGAGCTTACTCGGAGGTTTGAAGAAAAACTAGAGGAATTAGAAAAAGTTTCCAGGGATCCCAGCAATGAGAATCCTAAACTAAG
heAs pG l uTh r G l uAr gG l uLeuTh r A r gAr gPh eG l uG l uLy s LeuG l uG l uLeuG l uLy s Va l Se r A r gAs pP r oSe r A s nG l uAs nP r oLy s LeuAr
AGACCTCTACTTGGTCTTACAAGAAGAGTACCACTTAAAGCCAGAGACCAAGACCATTCTCTTCGTGAAGACCAGAGCACTCGTGGATGCTCTGAAGAAA
gAs pLeuT y r LeuVa l LeuG l nG l uG l uT y r Hi s LeuLy s P r oG l uTh r Ly s Th r I l eLeuPh eVa l Ly s Th r A r gA l a LeuVa l As pA l a LeuLy s Ly s
TGGATTGAAGAAAATCCTGCACTAAGCTTTCTAAAGCCTGGCATACTGACTGGGCGTGGCAGAACAAACCGGGCAACAGGAATGACGCTCCCGGCACAGA
T r p I l eG l uG l uAs nP r oA l a LeuSe r Ph eLeuLy s P r oG l y I l eLeuTh r G l yA r gG l yA r gTh r A s nAr gA l a Th r G l yMe t Th r LeuP r oA l a G l nL
AGTGTGTGCTGGAGGCATTCAGAGCCAGCGGAGATAACAATATTCTGATTGCTACCTCGGTCGCTGATGAAGGCATTGACATTGCTGAGTGCAATCTCGT
y s Cys Va l LeuG l uA l a Ph eAr gA l a Se r G l yAs pAs nAs n I l eLeu I l eA l a Th r Se r Va l A l aAs pG l uG l y I l eAs p I l eA l a G l uCysAs nLeuVa
CATTCTCTATGAGTACGTGGGCAACGTCATCAAGATGATCCAAACCAGAGGCCGAGGAAGAGCACGAGATAGCAAGTGCTTCCTCCTGACCAGCAGCGCT
l I l eLeuT y r G l uT y r Va l G l yAs nVa l I l eLy s Me t I l eG l nTh r A r gG l yA r gG l yA r gA l a A r gAs pSe r Ly s Cys Ph eLeuLeuTh r Se r Se r A l a
GACGTGATTGAAAAAGAAAAGGCGAACATGATCAAGGAAAAAATAATGAATGAATCCATCTTAAGACTGCAGACATGGGATGAAATGAAATTTGGAAAGA
As pVa l I l eG l uLy s G l uLysA l aAs nMe t I l eLy s G l uLy s I l eMe tAs nG l uSe r I l eLeuAr gLeuG l nTh r T r pAs pG l uMe t Ly s Ph eG l yL y s T
CGGTTCACCGCATACAGGTGAATGAAAAACTCCTCAGAGACAGTCAGCACAAACCACAACCTGTTCCTGACAAAGAAAACAAGAAACTGCTGTGTGGAAA
hr Va l Hi s A r g I l eG l nVa l As nG l uLy s LeuLeuAr gAs pSe r G l nHi s Ly s P r oG l nP r oVa l P r oAs pLy s G l uAs nLy s Ly s LeuLeuCys G l yL y
GTGCAAGAATTTTGCGTGCTACACAGCTGACATTCGAGTGGTTGAGACGTCCCACTACACTGTCCTTGGAGACGCTTTTAAGGAGCGCTTTGTGTGTAAG
s Cys LysAs nPh eA l a Cys T y r Th r A l aAs p I l eAr gVa l Va l G l uTh r Se r Hi s T y r Th r Va l LeuG l yAs pA l a Ph eLy s G l uAr gPh eVa l Cys Ly s
3101
847
3201
880
CCACACCCTAAACCAAAGATCTATGACAATTTTGAGAAGAAAGCAAAGATATTCTGCGCCAAACAGAACTGTAGCCACGACTGGGGAATTTTTGTGAGAT
P r oHi s P r oLy s P r oLy s I l eTyrAs pAs nPh eG l uLy s LysA l a Ly s I l ePh eCysA l a Ly s G l nAs nCys Se r Hi sAs pT r pG l y I l ePh eVa l A r gT
ACAAGACGTTCGAGATTCCAGTCATAAAAATTGAAAGTTTCGTCGTGGAAGATATTGTGAGCGGAGTTCAGAACCGGCACTCAAAGTGGAAGGACTTTCA
y r Ly s Th r Ph eG l u I l eP r oVa l I l eLy s I l eG l uSe r Ph eVa l Va l G l uAs p I l eVa l Se r G l yVa l G l nAs nAr gHi s Se r Ly s T r pLysAs pPh eHi
BglII (3117)
XbaI (3364)
3301 TTTTGAAAGGATACAGTTCGATCCTGCAGAAATGTCCGTATGACCTCAGGCTTCTCCGTCTCGTCTAGACCTAGCTCGACATGATAAGATACATTGATGA
913 s Ph eG l uAr g I l eG l nPh eAs pP r oA l a G l uMe t Se r Va l •••
3401 GTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTGAAATTTGTGATGCTATTGCTT
HpaI (3535)
3501 TATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAG
PacI (3644)
SwaI (3634)
BspLU11I (3650)
3601 CAAGTAAAACCTCTACAAATGTGGTAGATCATTTAAATGTTAATTAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGT
3701 TGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCA
3801 GGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTT
3901 TCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCT
4001 TATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGG
4101 CGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAA
4201 AGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAG
PacI (4384)
4301 AAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGGCTAGTTAATTAAGCTGTACACTGTG
4401 GAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAG
4501 TCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTC
NcoI (4626)
4601 CGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTG
AvrII (4719)
SmaI (4742)
BbrPI (4779) AseI (4790)
4701 AGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATcagCACGTGTTGACAATTAATCATCGG
XmnI (4854)
4801
4901
18
5001
52
5101
85
5201
118
5301
5401
BspHI (4846)
CATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAATCATGAAGACCTTCAACATCTCTCAGCAGGATCTGGAGCTGGTGGAGGTCGCCAC
1 Me t Ly s Th r Ph eAs n I l eSe r G l nG l nAs pLeuG l uLeuVa l G l uVa l A l a Th
TGAGAAGATCACCATGCTCTATGAGGACAACAAGCACCATGTCGGGGCGGCCATCAGGACCAAGACTGGGGAGATCATCTCTGCTGTCCACATTGAGGCC
r G l uLy s I l eTh r Me t LeuT y r G l uAs pAs nLy s Hi s Hi s Va l G l yA l aA l a I l eAr gTh r Ly s Th r G l yG l u I l e I l eSe r A l a Va l Hi s I l eG l uA l a
TACATTGGCAGGGTCACTGTCTGTGCTGAAGCCATTGCCATTGGGTCTGCTGTGAGCAACGGGCAGAAGGACTTTGACACCATTGTGGCTGTCAGGCACC
T y r I l eG l yA r gVa l Th r Va l CysA l a G l uA l a I l eA l a I l eG l ySe r A l a Va l Se r A s nG l yG l nLysAs pPh eAs pTh r I l eVa l A l a Va l A r gHi s P
CCTACTCTGATGAGGTGGACAGATCCATCAGGGTGGTCAGCCCCTGTGGCATGTGCAGAGAGCTCATCTCTGACTATGCTCCTGACTGCTTTGTGCTCAT
r oT y r Se r A s pG l uVa l As pAr gSe r I l eAr gVa l Va l Se r P r oCys G l yMe t CysAr gG l uLeu I l eSe r A s pT y r A l a P r oAs pCys Ph eVa l Leu I l
EcoRI (5275)
TGAGATGAATGGCAAGCTGGTCAAAACCACCATTGAGGAACTCATCCCCCTCAAGTACACCAGGAACTAAACCTGAATTCGCTAGAGGGCCCTATTCTAT
eG l uMe tAs nG l yL y s LeuVa l Ly s Th r Th r I l eG l uG l uLeu I l eP r oLeuLy s T y r Th r A r gAs n•••
AGTGTCACCTAAATGCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCT
GGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAG
NotI (5562)
XhoI (5556)
5501 GACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCGCAGGGCCCAATTGCTCGAGCGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTG
5601 TGTTGGTTTTTTGTGTGAATCGTAACTAACATACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCA
5701 GGTGCCAGAACATTTCTCTATCGAA