LOCUS Exported 5340 bp ds-DNA circular SYN 09-DEC-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pET His6 TEV LIC cloning vector (1B) SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5340) TITLE LIC N-terminal fusion vectors for E. coli expression REFERENCE 2 (bases 1 to 5340) AUTHORS . TITLE Direct Submission JOURNAL Exported Dec 9, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5340 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind 959..978 /label=LacI-R /note="LacI, reverse primer" promoter complement(998..1075) /gene="lacI" /label=lacI promoter primer_bind 1281..1300 /label=pBRrevBam /note="pBR322 vectors, tet region, downstream of BamHI, reverse primer" primer_bind 1384..1403 /label=T7 /note="T7 promoter, forward primer" promoter 1384..1402 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" protein_bind 1403..1427 /label=lac operator /bound_moiety="lac repressor encoded by 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)." RBS 1442..1464 /note="efficient ribosome binding site from bacteriophage T7 gene 10 (Olins and Rangwala, 1989)" CDS 1483..1500 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" CDS 1501..1521 /codon_start=1 /product="tobacco etch virus (TEV) protease recognition and cleavage site" /label=TEV site /translation="ENLYFQS" CDS 1587..1604 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" primer_bind complement(1657..1675) /label=T7 Term /note="T7 terminator, reverse primer" terminator 1671..1718 /label=T7 terminator /note="transcription terminator for bacteriophage T7 RNA polymerase" rep_origin 1755..2210 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(1842..1861) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2052..2073 /label=F1ori-F /note="F1 origin, forward primer" CDS complement(2303..3118) /codon_start=1 /gene="aph(3')-Ia" /product="aminoglycoside phosphotransferase" /label=KanR /note="confers resistance to kanamycin in bacteria or G418 (Geneticin(R)) in eukaryotes" /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF" primer_bind 3026..3045 /label=Kan-R /note="Kanamycin resistance gene, reverse primer" primer_bind 3110..3129 /label=pENTR-R /note="pENTR vectors, reverse primer" rep_origin 3240..3828 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 3729..3748 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 3982..3999 /label=L4440 /note="L4440 vector, forward primer" misc_feature 4014..4153 /label=bom /note="basis of mobility region from pBR322" primer_bind complement(4079..4098) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" primer_bind 4239..4261 /label=pGEX 3' /note="pGEX vectors, reverse primer" CDS complement(4255..4446) /codon_start=1 /gene="rop" /product="Rop protein, which maintains plasmids at low copy number" /label=rop /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA DELYRSCLARFGDDGENL" CDS complement(5255..997) /codon_start=1 /gene="lacI" /product="lac repressor" /label=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" ORIGIN 1 tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga ttgcccttca 61 ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa 121 aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg gtatcgtcgt 181 atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg 241 cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgata ccctcattca 301 gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc cgttccgcta 361 tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc agacgcgccg 421 agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat gcgaccagat 481 gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg atgggtgtct 541 ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc acagcaatgg 601 catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc gcgagaagat 661 tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac accaccacgc 721 tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca 781 gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc agttgttgtg 841 ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt tcccgcgttt 901 tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa gagacaccgg 961 catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg aattgactct 1021 cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg gtgtccggga 1081 tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag taggttgagg 1141 ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc 1201 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg 1261 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg 1321 gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga 1381 aattaatacg actcactata ggggaattgt gagcggataa caattcccct ctagaaataa 1441 ttttgtttaa ctttaagaag gagatatacc atgggttctt ctcaccatca ccatcaccat 1501 gaaaacctgt acttccaatc caatattgga agtggataac ggatccgaat tcgagcgccg 1561 tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat ccggctgcta 1621 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac 1681 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg 1741 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 1801 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 1861 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 1921 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 1981 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 2041 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta 2101 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 2161 ttaacaaaaa tttaacgcga attttaacaa actagtaacg tttacaattt caggtggcac 2221 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 2281 gtatccgctc atgaattaat tcttagaaaa actcatcgag catcaaatga aactgcaatt 2341 tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag 2401 aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga 2461 ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg 2521 agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagttta tgcatttctt 2581 tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc gcatcaacca 2641 aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag 2701 gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa 2761 tgttttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc ccggggatcg 2821 cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg gtcggaagag 2881 gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca ttggcaacgc 2941 tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac aatcgataga 3001 ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat aaatcagcat 3061 ccatgttgga atttaatcgc ggcctagagc aagacgtttc ccgttgaata tggctcataa 3121 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgac caaaatccct 3181 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 3241 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 3301 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 3361 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 3421 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 3481 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 3541 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 3601 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 3661 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 3721 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 3781 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 3841 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 3901 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 3961 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg 4021 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta 4081 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg 4141 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 4201 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 4261 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc 4321 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag 4381 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt 4441 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa 4501 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg 4561 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg 4621 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc 4681 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta 4741 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca 4801 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc 4861 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggggccgc 4921 catgccggcg ataatggcct gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa 4981 ggcttgagcg agggcgtgca agattccgaa taccgcaagc gacaggccga tcatcgtcgc 5041 gctccagcga aagcggtcct cgccgaaaat gacccagagc gctgccggca cctgtcctac 5101 gagttgcatg ataaagaaga cagtcataag tgcggcgacg atagtcatgc cccgcgccca 5161 ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc ccggtgccta 5221 atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 5281 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat //