pET100/D-TOPO

LOCUS       pET100/D-TOPO               5764 bp ds-DNA     circular SYN 15-AUG-2016
DEFINITION  synthetic circular DNA
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5764)
  AUTHORS   Biofeng Lab.
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..5764
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        209..227
                     /note="T7 promoter"
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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)."
     RBS             282..288
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     misc_feature    297..299
                     /note="ATG"
                     /note="Initiation ATG:"
     CDS             309..326
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /note="6xHis"
                     /translation="HHHHHH"
     CDS             330..362
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10
                     T7 tag (gene 10 leader)"
                     /note="T7 tag"
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             366..389
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /note="Xpress(TM) tag"
                     /translation="DLYDDDDK"
     misc_feature    396..400
                     /note="TOPO site1"
                     /note="TOPO recognition site1"
     misc_feature    405..409
                     /note="TOPO site 2"
                     /note="TOPO recognition site 2"
     terminator      427..555
                     /note="T7 terminator"
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(705..733)
                     /note="tet promoter"
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     promoter        856..954
                     /gene="bla"
                     /note="bla promoter"
     CDS             955..1815
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="AmpR"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      2022..2757
                     /direction=RIGHT
                     /note="pBR322"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    2760..2899
                     /note="bom"
                     /note="basis of mobility region from pBR322"
     CDS             complement(3001..3192)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /note="rop"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4507..5595)
                     /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="MVNVKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAM
                     AELNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVER
                     SGVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGD
                     WSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTED
                     SSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTA
                     SPRALADSLMQLARQVSRLESGQ"
     promoter        complement(5599..5664)
                     /gene="lacI"
                     /note="lacI promoter"
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc
      301 ggggttctca tcatcatcat catcatggta tggctagcat gactggtgga cagcaaatgg
      361 gtcgggatct gtacgacgat gacgataagg atcatccctt caccaagggc gagctcaacg
      421 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
      481 aactagcata accccttggg gcctctaaac gggtcttgag gagttttttg ctgaaaggag
      541 gaactatatc cggatatccc gcaagaggcc cggcagtacc ggcataacca agcctatgcc
      601 tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag atttcataca
      661 cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa gcttatcgat
      721 gataagctgt caaacatgag aattaattct tgaagacgaa agggcctcgt gatacgccta
      781 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
      841 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      901 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      961 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     1021 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     1081 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     1141 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     1201 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1261 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1321 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1381 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1441 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     1501 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1561 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1621 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1681 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1741 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1801 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1861 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1921 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1981 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2041 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2101 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     2161 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     2221 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2281 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2341 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2401 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2461 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2521 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2581 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2641 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2701 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2761 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     2821 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     2881 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     2941 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     3001 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     3061 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     3121 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     3181 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     3241 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     3301 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     3361 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     3421 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     3481 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     3541 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     3601 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     3661 ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat
     3721 ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc
     3781 tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag
     3841 gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg
     3901 cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg
     3961 atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt
     4021 ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg
     4081 ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc
     4141 agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg
     4201 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     4261 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     4321 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     4381 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     4441 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     4501 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4561 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     4621 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     4681 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     4741 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     4801 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     4861 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     4921 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     4981 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     5041 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     5101 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     5161 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     5221 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     5281 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     5341 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     5401 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     5461 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
     5521 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
     5581 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
     5641 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
     5701 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
     5761 catg
//