pET-41 Ek/LIC
At a Glance
pET-41 Ek/LIC, Image made with PlasMapper
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| Type |
Cloning/Expression |
|---|---|
| Origin of Replication |
f1, pBR322 |
| Copy # |
~40 |
| Markers |
Kanamycin |
| Link to Sequence | |
| Features |
T7 Promoter |
| Accession Number |
|
| edit table |
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Notes
pET-41 is used for rapid, directional cloning of PCR-amplified DNA for high level expression of polypeptides fused with N-terminal GST•Tag, His•Tag, and S•Tag sequences. Using specifically designed primers for amplification, inserts can be efficiently cloned without the need for restriction digestion or ligation.
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 tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
121 tgtttagcag cctaggtatt aatcaattag tggtggtggt ggtggtggtg gtgctcgagt
181 gcggccgcaa gcttgtcgac ggagctcgcc tgcaggcgcg ccaaggcctg tacagaattc
241 ggatccccga tatccatggt tgaggagaag cccgggctct tgtcgtcgtc atcaccagaa
301 ccaccaccgg tacccagatc tgggctgtcc atgtgctggc gttcgaattt agcagcagcg
361 gtttctttca taccaattgc agtactaccg cgtggcacca gacccgcgga gtgatggtga
421 tggtgatgac cagaaccact agttgaacca tccgattttg gaggatggtc gccaccacca
481 aacgtggctt gccagccctg caaaggccat gctatatact tgctggattt caagtactta
541 tcaatttgtg ggatagcttc aatacgtttt ttaaaacaaa ctaattttgg gaacgcatcc
601 aggcacattg ggtccatgta taaaacaaca tcaagagcgt catacaacat gaagtcagga
661 tgggttacat gatcaccatt taaatatgtt ttatgacata aacgatcttc gaacattttc
721 agcatttcag gtagcttgct aagaaaatca actttgagag tttcaaagtc tttactatat
781 gcaattctcg aaacaccgta tctaatatcc aaaaccgctc cttcaagcat tgaaatctct
841 gcacgctctt ttggacaacc acccaacatg ttgtgcttgt cagctatata acgtatgatg
901 gccatagact gtgttaattt aacatcacca tcaatataat aaggaagatt gggaaactcc
961 aaacccaatt caaacttttt gtttcgccat ttatcacctt catcgcgctc atacaaatgc
1021 tcttcatatt tttcttcaag atattccaaa agaagtcgag tgggttgcac aaggccctta
1081 attttccaat aacctagtat aggggacata tgtatatctc cttcttaaag ttaaacaaaa
1141 ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt
1201 tcgcgggatc gagatcgatc tcgatcctct acgccggacg catcgtggcc ggcatcaccg
1261 gcgccacagg tgcggttgct ggcgcctata tcgccgacat caccgatggg gaagatcggg
1321 ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg tatggtggca ggccccgtgg
1381 ccgggggact gttgggcgcc atctccttgc atgcaccatt ccttgcggcg gcggtgctca
1441 acggcctcaa cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
1501 gagatcccgg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
1561 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
1621 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
1681 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
1741 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
1801 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
1861 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
1921 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
1981 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
2041 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
2101 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
2161 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
2221 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
2281 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
2341 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggacat ctcggtagtg
2401 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
2461 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
2521 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
2581 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
2641 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
2701 tcattaggca ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt
2761 ccggtgggcg cggggcatga ctagcatgat cgtgctcctg tcgttgagga cccggctagg
2821 ctggcggggt tgccttactg gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag
2881 cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca tgaatggtct tcggtttccg
2941 tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc accattatgt tccggatctg
3001 catcgcagga tgctgctggc taccctgtgg aacacctaca tctgtattaa cgaagcgctg
3061 gcattgaccc tgagtgattt ttctctggtc ccgccgcatc cataccgcca gttgtttacc
3121 ctcacaacgt tccagtaacc gggcatgttc atcatcagta acccgtatcg tgagcatcct
3181 ctctcgtttc atcggtatca ttacccccat gaacagaaat cccccttaca cggaggcatc
3241 agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc tttatcagaa gccagacatt
3301 aacgcttctg gagaaactca acgagctgga cgcggatgaa caggcagaca tctgtgaatc
3361 gcttcacgac cacgctgatg agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg
3421 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
3481 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
3541 catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc ggcatcagag
3601 cagattgtac tgagagtgca ccatatatgc ggtgtgaaat accgcacaga tgcgtaagga
3661 gaaaataccg catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
3721 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
3781 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
3841 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
3901 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
3961 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
4021 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
4081 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
4141 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
4201 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
4261 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
4321 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
4381 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
4441 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
4501 actcacgtta agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat
4561 acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat
4621 tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca
4681 ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat
4741 ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg
4801 gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta
4861 ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca
4921 ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt
4981 tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg
5041 aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa
5101 caagtctgga aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat
5161 ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat
5221 gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc
5281 ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct
5341 gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attaattcat
5401 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
5461 tccccgaaaa gtgccacctg aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa
5521 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
5581 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
5641 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
5701 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
5761 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
5821 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
5881 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
5941 ttcgcca
