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Plasmid Files

pGGA

Cloning vector with two BsaI restriction sites for Golden Gate assembly.

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pGGA.dna
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Sequence Author:  New England Biolabs
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AflIII - PciI (2054) DrdI (1952) BssS α I (1881) HaeII (1814) PspFI (1754) BseYI (1750) BsiHKAI (1744) ApaLI (1740) AlwNI (1645) AcuI (1512) BsaAI (1299) Bpu10I (1216) TsoI (1093) PvuII (1092) Forward (CW) Analysis Primer (234 .. 260) PstI - SbfI (238) AarI (241) PmeI (247) PspXI - XhoI (284) BamHI (290) EcoRI (296) BstBI (300) BsaI (314) BglII (333) BsaI (355) PacI (377) BsaHI (382) ZraI (383) AatII (385) EcoRI (388) XhoI (393) EagI - NotI (400) AseI (434) Cloning Analysis Reverse Primer (411 .. 436) BanI (522) TatI (573) ScaI (575) SspI (680) BtgI - NcoI - StyI (687) MscI (725) PasI (757) PflMI * (763) BpmI (872) EcoRI (988) BspEI (992) pGGA 2174 bp
AflIII  (2054)
1 site
A C R Y G T T G Y R C A

Sticky ends from different AflIII sites may not be compatible.
PciI  (2054)
1 site
A C A T G T T G T A C A

PciI is inhibited by nonionic detergents.
DrdI  (1952)
1 site
G A C N N N N N N G T C C T G N N N N N N C A G

Sticky ends from different DrdI sites may not be compatible.
BssSαI  (1881)
1 site
C A C G A G G T G C T C
HaeII  (1814)
1 site
R G C G C Y Y C G C G R
PspFI  (1754)
1 site
C C C A G C G G G T C G
BseYI  (1750)
1 site
C C C A G C G G G T C G

After cleavage, BseYI can remain bound to DNA and alter its electrophoretic mobility.
BsiHKAI  (1744)
1 site
G W G C W C C W C G W G

Sticky ends from different BsiHKAI sites may not be compatible.
ApaLI  (1740)
1 site
G T G C A C C A C G T G
AlwNI  (1645)
1 site
C A G N N N C T G G T C N N N G A C

Sticky ends from different AlwNI sites may not be compatible.
AcuI  (1512)
1 site
C T G A A G ( N ) 14 N N G A C T T C ( N ) 14

Efficient cleavage requires at least two copies of the AcuI recognition sequence.
Sticky ends from different AcuI sites may not be compatible.
After cleavage, AcuI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
BsaAI  (1299)
1 site
Y A C G T R R T G C A Y
Bpu10I  (1216)
1 site
C C T N A G C G G A N T C G

Efficient cleavage requires at least two copies of the Bpu10I recognition sequence.
This recognition sequence is asymmetric, so ligating sticky ends generated by Bpu10I will not always regenerate a Bpu10I site.
Sticky ends from different Bpu10I sites may not be compatible.
TsoI  (1093)
1 site
T A R C C A ( N ) 9 N N A T Y G G T ( N ) 9

Sticky ends from different TsoI sites may not be compatible.
After cleavage, TsoI can remain bound to DNA and alter its electrophoretic mobility.
For full activity, add fresh S-adenosylmethionine (SAM).
PvuII  (1092)
1 site
C A G C T G G T C G A C
PstI  (238)
1 site
C T G C A G G A C G T C
SbfI  (238)
1 site
C C T G C A G G G G A C G T C C
AarI  (241)
1 site
C A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4

Efficient cleavage requires at least two copies of the AarI recognition sequence.
Sticky ends from different AarI sites may not be compatible.
After cleavage, AarI can remain bound to DNA and alter its electrophoretic mobility.
PmeI  (247)
1 site
G T T T A A A C C A A A T T T G
PspXI  (284)
1 site
V C T C G A G B B G A G C T C V
XhoI  (284)
2 sites
C T C G A G G A G C T C
BamHI  (290)
1 site
G G A T C C C C T A G G

After cleavage, BamHI-HF™ (but not the original BamHI) can remain bound to DNA and alter its electrophoretic mobility.
EcoRI  (296)
3 sites
G A A T T C C T T A A G
BstBI  (300)
1 site
T T C G A A A A G C T T
BsaI  (314)
2 sites
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
BglII  (333)
1 site
A G A T C T T C T A G A
BsaI  (355)
2 sites
G G T C T C N C C A G A G N ( N ) 4

Sticky ends from different BsaI sites may not be compatible.
BsaI can be used between 37°C and 50°C.
PacI  (377)
1 site
T T A A T T A A A A T T A A T T
BsaHI  (382)
1 site
G R C G Y C C Y G C R G

BsaHI is typically used at 37°C, but is even more active at 60°C.
ZraI  (383)
1 site
G A C G T C C T G C A G
AatII  (385)
1 site
G A C G T C C T G C A G
EcoRI  (388)
3 sites
G A A T T C C T T A A G
XhoI  (393)
2 sites
C T C G A G G A G C T C
EagI  (400)
1 site
C G G C C G G C C G G C
NotI  (400)
1 site
G C G G C C G C C G C C G G C G
AseI  (434)
1 site
A T T A A T T A A T T A
BanI  (522)
1 site
G G Y R C C C C R Y G G

Sticky ends from different BanI sites may not be compatible.
TatI  (573)
1 site
W G T A C W W C A T G W
ScaI  (575)
1 site
A G T A C T T C A T G A
SspI  (680)
1 site
A A T A T T T T A T A A
BtgI  (687)
1 site
C C R Y G G G G Y R C C

Sticky ends from different BtgI sites may not be compatible.
NcoI  (687)
1 site
C C A T G G G G T A C C
StyI  (687)
1 site
C C W W G G G G W W C C

Sticky ends from different StyI sites may not be compatible.
MscI  (725)
1 site
T G G C C A A C C G G T
PasI  (757)
1 site
C C C W G G G G G G W C C C

Sticky ends from different PasI sites may not be compatible.
PflMI  (763)
1 site
C C A N N N N N T G G G G T N N N N N A C C
* Blocked by Dcm methylation.
Sticky ends from different PflMI sites may not be compatible.
BpmI  (872)
1 site
C T G G A G ( N ) 14 N N G A C C T C ( N ) 14

Efficient cleavage requires at least two copies of the BpmI recognition sequence.
Sticky ends from different BpmI sites may not be compatible.
After cleavage, BpmI can remain bound to DNA and alter its electrophoretic mobility.
BpmI quickly loses activity at 37°C.
EcoRI  (988)
3 sites
G A A T T C C T T A A G
BspEI  (992)
1 site
T C C G G A A G G C C T
Forward (CW) Analysis Primer
27-mer  /  44% GC
1 binding site
234 .. 260  =  27 annealed bases
Tm  =  61°C
Cloning Analysis Reverse Primer
26-mer  /  46% GC
1 binding site
411 .. 436  =  26 annealed bases
Tm  =  58°C
CmR
547 .. 1206  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
CmR
547 .. 1206  =  660 bp
219 amino acids  =  25.7 kDa
Product: chloramphenicol acetyltransferase
confers resistance to chloramphenicol
ori
1410 .. 1998  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
ori
1410 .. 1998  =  589 bp
high-copy-number ColE1/pMB1/pBR322/pUC origin of replication
cat promoter
1207 .. 1309  =  103 bp
promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase
cat promoter
1207 .. 1309  =  103 bp
promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase
BsaI insert
315 .. 359  =  45 bp
fragment released by digestion with BsaI
BsaI insert
315 .. 359  =  45 bp
fragment released by digestion with BsaI
downstream MCS
373 .. 406  =  34 bp
downstream multiple cloning site
downstream MCS
373 .. 406  =  34 bp
downstream multiple cloning site
upstream MCS
283 .. 304  =  22 bp
upstream multiple cloning site
upstream MCS
283 .. 304  =  22 bp
upstream multiple cloning site
SP6 promoter
254 .. 272  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
SP6 promoter
254 .. 272  =  19 bp
promoter for bacteriophage SP6 RNA polymerase
T7 promoter
418 .. 436  =  19 bp
promoter for bacteriophage T7 RNA polymerase
T7 promoter
418 .. 436  =  19 bp
promoter for bacteriophage T7 RNA polymerase
ORF:  27 .. 263  =  237 bp
ORF:  78 amino acids  =  9.0 kDa
ORF:  547 .. 1206  =  660 bp
ORF:  219 amino acids  =  25.7 kDa
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