pET-43.1 Ek/LIC
At a Glance
pET-43.1 Ek/LIC, Image made with PlasMapper
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| Type |
Cloning/Expression |
|---|---|
| Origin of Replication |
pBR322, fl |
| Copy # |
~40 |
| Markers |
Ampicillin |
| Link to Sequence | |
| Features |
T7 Promoter |
| Accession Number |
|
| edit table |
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Notes
pET-43.1 Ek/LIC is prepared for rapid, directional cloning of PCR-amplified DNA for high-level expression of polypeptides fused with N-terminal Nus•Tag, His•Tag and S•Tag sequences.
History
Ligation-independent cloning (LIC) was developed for directional cloning of PCR products without restriction enzyme digestion or ligation reactions[1][2]. LIC takes advantage of the 3’→5’ exonuclease activity of T4 DNA polymerase to create very specific 12 to 15 nucleotide single-stranded overhangs in the vector and the insert, so that the vast majority of annealed products consist of the desired molecules. The annealed LIC vector and insert are transformed into competent E. coli cells, and covalent bonds are formed at the vector-insert junctions within the cell to yield circular plasmid. Directional cloning of the insert is achieved with minimal non-recombinant background, and cloning is so efficient that virtually all of the resulting colonies contain the desired recombinant.
Sources
References
See Help:References for how to manage references in EcoliWiki.
Sequence
1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
61 ggggttatgc tagttattgc tcagcggata catatttgaa tgtatttaga aaaataaaca
121 aaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc ttaatttgat
181 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcgcaacgt
241 tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
301 taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggcagttc
361 cctactctcg catggggaga ccccacacta ccatcggcgc tacggcgttt cacttctgag
421 ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaatt ctgttttatc
481 agcctaggaa cgcccaactt aattaacatt agtggtggtg gtggtggtgc tcgagatcct
541 ctgggtcttc aggagcgagt tctggctggc ttgcacgtgt atacagctgt gcggccgcaa
601 gcttcgaacg cgtatcgatg gtaccgtcga cgtcctgcag gcgcgcctgt acagaattcg
661 gatccacgag ctcagatatc gttgaggaga agcccggact cttgtcgtcg tcatcaatcg
721 taccagaacc cgcgctgccc cgggggacca gaccagttgg cggtggcgag tccatgtgct
781 ggcgttcaaa tttcgcagca gcggtttctt tacccgcgga gtgatggtga tggtgatgac
841 cagaaccact agtcgcttcg tcaccgaacc agcaaatatt acgggcagcc ataatcagtg
901 ctccggcttt ttcgtcggtc aacccttcga tatcagccag atcatcaatg ccctgttcgg
961 cgagatcttc cagcgtacaa acgccacggg cggccagttt gaatgccaaa tcacgatcta
1021 ccccttcaag gttcagcaga tcgtcagccg gtttgttatc accgaggctt tcttcctggg
1081 cctgtgcaat ggtggccagt gcatttttag cacgctcgcg cagtgcttca acggtcggct
1141 catcaaggcc ttcgatttcc aacagctctt tcatcggcac ataggccaat tcttccagcg
1201 tcgagaagcc ttcttctacc agaacagtcg cgaagtcttc gtcgatgtcg agatatttgg
1261 tgaaggtgtc gatcgctgcg tgcgcttccg cctgatgctt agcttgcagg tcgtcaacgg
1321 tcatcacgtt gagttcccaa ccgctcagtt gcgaagccag acgcacgttc tgaccgttac
1381 ggccaatcgc ctgcgccaga ttaccggctt caacggcgat gtccatggtg tgtttatctt
1441 catccaccac gatagaagca acgtctgccg gtgccattgc gttaatcacg aactgcgccg
1501 ggttatcatc ccacaggacg atatcgatac gctcgccacc cagttcagta gacaccgcct
1561 gaacacgcgc gccacgcata cctacgcaag cacctaccgg atcgatacgt ttatcgttgg
1621 ttttcaccgc gattttcgca cgagaacccg gatcgcgagc cgctgcttta atttcaatca
1681 cttcttcgcc gatttctggc acttcaatac ggaacagttc gatcagcatt tccggcttgg
1741 aacgagtgac gaacagttgc gcgccacgcg cttccgggcg aacggaatag agcacgccac
1801 gaacgcggtc gccagggcgg aagttttcac gcggcagcat atcttcgcgc aggatcacgg
1861 cttcagcgtt gttgcccaga tccagagaga tgttgtcgcg gtttactttt ttcaccacgc
1921 cggtgatgat ttcaccttcg tgttcacgga actgatcaac caccatcgca cgttcggctt
1981 cacgcacttt ctgcacgata acctgttttg ccgtctgggt agtgatacgg tcaaaggtaa
2041 cagactcaat ctgatcttca acgtaatcgc ccaggttcag gctttcatct tcataacgtg
2101 cggcttcaag ggtgatttcc ttggtcggct gggtgacttc atcaacaact aaccagcgac
2161 ggaaagtgtc aaaatcaccg cttttgcgat cgatctgtac gcggacgtcg atctcttgtt
2221 catatttttt ctttgttgct gtcgccagcg cgctttccaa tgcttcgaaa atcttctcgc
2281 gaggtagcgc cttttcattg gatacggctt caactacagc caaaatttct ttgttcatat
2341 gtatatctcc ttcttaaagt taaacaaaat tatttctaga ggggaattgt tatccgctca
2401 caattcccct atagtgagtc gtattaattt cgcgggatcg agatcgatct cgatcctcta
2461 cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat
2521 cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt
2581 cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca
2641 tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct
2701 aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa
2761 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
2821 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
2881 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
2941 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
3001 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
3061 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
3121 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
3181 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
3241 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
3301 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
3361 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
3421 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
3481 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
3541 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
3601 tgcgcgttgg tgcggacatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
3661 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
3721 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
3781 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
3841 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
3901 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc
3961 ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tagcatgatc
4021 gtgctcctgt cgttgaggac ccggctaggc tggcggggtt gccttactgg ttagcagaat
4081 gaatcaccga tacgcgagcg aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga
4141 gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca
4201 gcgccctgca ccattatgtt ccggatctgc atcgcaggat gctgctggct accctgtgga
4261 acacctacat ctgtattaac gaagcgctgg cattgaccct gagtgatttt tctctggtcc
4321 cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca
4381 tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca tcggtatcat tacccccatg
4441 aacagaaatc ccccttacac ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac
4501 atggcccgct ttatcagaag ccagacatta acgcttctgg agaaactcaa cgagctggac
4561 gcggatgaac aggcagacat ctgtgaatcg cttcacgacc acgctgatga gctttaccgc
4621 agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct gacacatgca gctcccggag
4681 acggtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca gggcgcgtca
4741 gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt cacgtagcga tagcggagtg
4801 tatactggct taactatgcg gcatcagagc agattgtact gagagtgcac catatatgcg
4861 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc
4921 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
4981 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
5041 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
5101 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
5161 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
5221 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
5281 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
5341 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
5401 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
5461 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
5521 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
5581 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
5641 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
5701 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgaccta
5761 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
5821 aaatgcttca atcatgattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
5881 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
5941 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
6001 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
6061 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
6121 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
6181 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
6241 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
6301 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
6361 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
6421 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
6481 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
6541 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
6601 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
6661 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
6721 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
6781 atctaggtga agatcctttt tgataatctc atgccatcgt gccgccagaa attgtaaacg
6841 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat
6901 aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
6961 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc
7021 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt
7081 tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
7141 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg
7201 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc
7261 ttaatgcgcc gctacagggc gcgtcccatt cgcca
