LOCUS       sequence-450923-        8081 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of recombinant Escherichia coli topoisomerase I.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8081)
  AUTHORS   Tan K, Zhou Q, Cheng B, Zhang Z, Joachimiak A, Tse-Dinh YC
  TITLE     Structural basis for suppression of hypernegative DNA supercoiling 
            by E. coli topoisomerase I.
  JOURNAL   Nucleic Acids Res. 2015 Dec 15;43(22):11031-46. doi: 
            10.1093/nar/gkv1073. Epub 2015 Oct 20.
  PUBMED    26490962
REFERENCE   2  (bases 1 to 8081)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1093/nar/gkv1073"; journalName: "Nucleic Acids Res"; date: 
            "2015-12-15- 15"; volume: "43"; issue: "22"; pages: "11031-46"
COMMENT     Sequence Label: pLIC-EcTOP
FEATURES             Location/Qualifiers
     source          1..8081
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    775..914
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(840..859)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1000..1022
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1016..1207)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2016..3098)
                     /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"
     primer_bind     3060..3079
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3099..3176)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3382..3401
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3485..3504
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3485..3503
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3504..3528
                     /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             3543..3565
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3584..3601
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3602..3622
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             6429..6446
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6499..6517)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      6513..6560
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      6597..7052
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6684..6703)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6894..6915
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(7145..7960)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(7145..7960)
                     /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     7868..7887
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7952..7971
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
      781 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
      841 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
      901 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      961 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     1021 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     1081 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     1141 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     1201 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     1261 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     1321 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     1381 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     1441 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     1501 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     1561 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     1621 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg
     1681 ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga
     1741 aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg
     1801 cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta
     1861 cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc
     1921 accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct
     1981 aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     2041 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     2101 ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc
     2161 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2221 aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg
     2281 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2341 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat accctcattc
     2401 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2461 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2521 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2581 tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc
     2641 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2701 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2761 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2821 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2881 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2941 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     3001 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     3061 gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc
     3121 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg
     3181 atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag
     3241 gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc
     3301 cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg
     3361 gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt
     3421 ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg
     3481 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc tctagaaata
     3541 attttgttta actttaagaa ggagatatac catgggttct tctcaccatc accatcacca
     3601 tgaaaacctg tacttccaat ccaatgcagg taaagctctt gtcatcgttg agtccccggc
     3661 aaaagccaaa acgatcaaca agtatctggg tagtgactac gtggtgaaat ccagcgtcgg
     3721 tcacatccgc gatttgccga ccagtggctc agctgccaaa aagagtgccg actctacctc
     3781 caccaagacg gctaaaaagc ctaaaaagga tgaacgtggc gctctcgtca accgtatggg
     3841 ggttgacccg tggcacaatt gggaggcgca ctatgaagtg ttgcctggta aagagaaggt
     3901 cgtctctgaa ctgaaacaac tggctgaaaa agccgaccac atctatctcg caaccgacct
     3961 tgaccgcgaa ggggaagcca ttgcatggca cctgcgggaa gtgattgggg gtgatgatgc
     4021 gcgctatagc cgagtggtgt ttaacgaaat tactaaaaac gcgatccgcc aggcatttaa
     4081 caaaccgggt gagctgaata ttgatcgtgt taatgcccag caggcgcgtc gctttatgga
     4141 ccgcgtggtg gggtatatgg tttcgccgct gctatggaaa aagatcgctc gtggcctgtc
     4201 tgccggtcgt gtgcagtcgg tggcggttcg cctggtggtc gagcgtgagc gtgaaattaa
     4261 agcgttcgtg ccggaagagt tctgggaagt cgatgccagc acgaccacgc catctggtga
     4321 agcgttggcg ttacaggtga ctcatcagaa cgacaaaccg ttccgtccgg tcaacaaaga
     4381 acaaactcag gctgcggtaa gtctgctgga aaaagcgcgc tacagcgtgc tggaacgtga
     4441 agacaaaccg acaaccagta aacctggcgc tccttttatt acctctacgc tgcaacaagc
     4501 tgccagcacc cgtcttggat ttggcgtgaa aaaaaccatg atgatggcgc agcgtttgta
     4561 tgaagcaggc tatatcactt acatgcgtac cgactccact aacctgagtc aggacgcggt
     4621 aaatatggtt cgcggttata tcagcgataa ttttggtaag aaatatctgc cggaaagtcc
     4681 gaatcagtac gccagcaaag aaaactcaca ggaagcgcac gaagcgattc gtccttctga
     4741 cgtcaatgtg atggcggaat cgctgaagga tatggaagca gatgcgcaga aactgtacca
     4801 gttaatctgg cgtcagttcg ttgcctgcca gatgacccca gcgaaatatg actccacgac
     4861 gctgaccgtt ggtgcgggcg atttccgcct gaaagcacgc ggtcgtattt tgcgttttga
     4921 tggctggaca aaagtgatgc ctgcgttgcg taaaggcgat gaagatcgca tcttaccagc
     4981 agttaataaa ggcgatgctc tgacgctcgt tgaacttaca ccagcccagc actttaccaa
     5041 gccgccagcc cgtttcagtg aagcatcgct ggttaaagag ctggaaaaac gcggtatcgg
     5101 tcgtccgtct acctatgcgt cgatcatttc gaccattcag gatcgtggct acgtgcgagt
     5161 agaaaatcgt cgtttctatg cggaaaaaat gggcgaaatc gtcaccgatc gccttgaaga
     5221 aaatttccgc gagttaatga actacgactt taccgcgcag atggaaaaca gcctcgacca
     5281 ggtggcaaat cacgaagcag agtggaaagc tgtactggat cacttcttct cggatttcac
     5341 ccagcagtta gataaagctg aaaaagatcc ggaagagggt ggtatgcgcc cgaaccagat
     5401 ggttctgacc agcattgact gcccgacttg tggtcgcaaa atggggattc gcacagcgag
     5461 caccggggta ttccttggct gttctggcta tgcgctgccg ccgaaagagc gttgcaaaac
     5521 caccattaac ctggtgccgg aaaacgaagt gctgaacgtg ctggaaggcg aagatgctga
     5581 aaccaacgcg ctgcgcgcaa aacgtcgttg cccgaaatgc ggcacggcga tggacagcta
     5641 tctcatcgat ccgaaacgta agttgcatgt ctgtggtaat aacccaacct gcgacggtta
     5701 cgagatcgaa gagggcgaat tccgcattaa aggttatgac ggcccgatcg ttgagtgtga
     5761 aaaatgtggc tctgaaatgc acctgaaaat ggggcgtttc ggtaaataca tggcctgcac
     5821 caacgaagag tgtaaaaaca cacgtaagat tttacgtaac ggcgaagtgg caccaccgaa
     5881 agaagatccg gtgccattac ctgagctgcc gtgcgaaaaa tcagatgctt atttcgtgct
     5941 gcgtgacggt gctgccggtg tgttcctggc tgccaacact ttcccgaaat cgcgtgaaac
     6001 gcgtgcgcca ctggtggaag agctttatcg cttccgcgac cgtctgccgg aaaaactgcg
     6061 ttatctggcc gatgcgccac agcaggatcc ggaaggtaat aagaccatgg ttcgctttag
     6121 ccgtaaaacc aaacagcaat atgtctcttc ggaaaaagac ggaaaggcga ctggctggtc
     6181 agcattttat gttgatggca aatgggttga aggaaaaaaa taacctttaa ttctgtcagg
     6241 tttttataaa caaagggtcg cgaaagcggc ccttttttat tgcatattat tttttcttca
     6301 cacctataca ctaaggctat aaatgatata gtggttatag ttagcacctt ttttattatt
     6361 aaatcgtatt agtcacccgc caggtgtgac gaaaaaacga tgttcccaag cttgcggccg
     6421 cactcgagca ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag
     6481 ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac
     6541 gggtcttgag gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc
     6601 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     6661 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     6721 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     6781 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     6841 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     6901 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     6961 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     7021 gaattttaac aaactagtaa cgtttacaat ttcaggtggc acttttcggg gaaatgtgcg
     7081 cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta
     7141 attcttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat
     7201 caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt
     7261 tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac
     7321 aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga
     7381 cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag
     7441 gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg
     7501 attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa
     7561 tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatgttttca cctgaatcag
     7621 gatattcttc taatacctgg aatgctgttt tcccggggat cgcagtggtg agtaaccatg
     7681 catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc
     7741 agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
     7801 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
     7861 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
     7921 gcggcctaga gcaagacgtt tcccgttgaa tatggctcat aacacccctt gtattactgt
     7981 ttatgtaagc agacagtttt attgttcatg accaaaatcc cttaacgtga gttttcgttc
     8041 cactgagcgt cagaccccgt agaaaagatc aaaggatctt c
//
