pET-41Ek/LIC

LOCUS       pET-41 Ek_LIC                5947 bp ds-DNA     circular SYN 23-OCT-2018
DEFINITION  Bacterial vector for ligation-independent cloning (LIC) to express 
            GST-tagged proteins with an enterokinase site.
ACCESSION   .
VERSION     .
KEYWORDS    pET-41 Ek_LIC
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5947)
  AUTHORS   MilliporeSigma (Novagen)
  TITLE     Direct Submission
  JOURNAL   Exported Saturday, June 20, 2020 from SnapGene Viewer 3.1.4
            http://www.snapgene.com
COMMENT     For LIC, linearize with BseRI and treat with T4 DNA polymerase plus 
            dTTP.
FEATURES             Location/Qualifiers
     source          1..5947
                     /organism="synthetic DNA construct"
                     /lab_host="Escherichia coli"
                     /mol_type="other DNA"
     terminator      26..73
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(150..173)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /note="8xHis"
                     /translation="HHHHHHHH"
     misc_feature    174..259
                     /note="MCS"
                     /note="multiple cloning site"
     CDS             complement(279..293)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /note="enterokinase site"
                     /translation="DDDDK"
     CDS             complement(324..368)
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /note="S-Tag"
                     /translation="KETAAAKFERQHMDS"
     CDS             complement(384..401)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /note="thrombin site"
                     /translation="LVPRGS"
     CDS             complement(411..428)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             complement(456..1109)
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /note="GST"
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     RBS             1118..1123
                     /note="ribosome binding site"
     protein_bind    1154..1178
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="lac operator"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(1179..1197)
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        1510..1587
                     /gene="lacI"
                     /note="lacI promoter"
     CDS             1588..2670
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /note="lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     CDS             3242..3433
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     rep_origin      complement(3863..4451)
                     /direction=LEFT
                     /note="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             4573..5388
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /note="KanR"
                     /note="confers resistance to kanamycin"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     rep_origin      complement(5481..5936)
                     /direction=LEFT
                     /note="f1 ori"
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        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
//