LOCUS       sequence-336816-        5849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To amplify transcription template containing LacI operators .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5849)
  AUTHORS   Xu W, Yan Y, Artsimovitch I, Dunlap D, Finzi L
  TITLE     Positive supercoiling favors transcription elongation through lac 
            repressor-mediated DNA loops.
  JOURNAL   Nucleic Acids Res. 2022 Mar 21;50(5):2826-2835. doi: 
            10.1093/nar/gkac093.
  PUBMED    35188572
REFERENCE   2  (bases 1 to 5849)
  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"; journalName: 
            "Nucleic Acids Res"; date: "2022-03-21- 21"; volume: "50"; issue: 
            "5"; pages: "2826-2835"
COMMENT     Sequence Label: pRS_1N_400
FEATURES             Location/Qualifiers
     source          1..5849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1027..1046
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1127..1144)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1298..1886)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1378..1397)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2057..2848,2849..2917))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2680..2699
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2918..3022)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3090..3108
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3135..3140,3141..3157,3158..3163)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             3211..4401
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(3563..3582)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             5040..5231
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5225..5247)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     protein_bind    5766..5782
                     /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)."
ORIGIN
        1 gcaaaacacc agcagcagtt tttccagttc cgtttatccg ggcaaaccat cgaagtgacc
       61 agcgaatacc tgttccgtca tagcgataac gagctcctgc actggatggt ggcgctggat
      121 ggtaagccgc tggcaagcgg tgaagtgcct ctggatgtcg ctccacaagg taaacagttg
      181 attgaactgc ctgaactacc gcagccggag agcgccgggc aactctggct cacagtacgc
      241 gtagtgcaac cgaacgcgac cgcatggtca gaagccgggc acatcagcgc ctggcagcag
      301 tagcgtctgg cggatcgatc gagctcaacg caattaaacc tcagtgtgac gctccccgcc
      361 gcgtcccacg ccatcccgca tctgaccacc agcgaaatgg atttttgcac tcgagctggg
      421 taataagcgt tggcaattta accgccagtc aggctttctt tcacagatgt ggattggcga
      481 taaaaaacaa ctgctgacgc cgctgcgcga tcagttcacc cgtgcaccgc tggataacga
      541 cattggcgta agtgaagcga cccgcattga ccctaacgcc tgggtcgaac gctggaaggc
      601 ggcgggccat taccaggccg aagcagcgtt gttgcagtgc acggcagata cacttgctga
      661 tgcggtgctg attacgaccg ctcacgcgtg gcagcatcag gggaaaacct tatttatcag
      721 ccggaaaacc taccggattg atggtagtgg tcaaatggcg attaccgttg atgttgaagt
      781 ggcgagcgat acaccgcatc cggcgcggat tggcctgaac tgccagctgg cgcaggtagc
      841 agagcgggta aactggctcg gattagcggc cgcaagaaaa ctatcccgac cgccttactg
      901 ccgcctgttt tgaccgctgg gatctgctgt aacagagcat tagcgcaagg tgatttttgt
      961 cttcttgcgc taattttttc cattgtctag agtagcgata gcggagtgta tactggctta
     1021 actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc
     1081 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     1141 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     1201 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     1261 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     1321 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     1381 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     1441 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     1501 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     1561 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     1621 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     1681 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     1741 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     1801 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     1861 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     1921 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     1981 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     2041 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2101 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     2161 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     2221 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     2281 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     2341 ttaatagttt gcgcaacgtt gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt
     2401 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     2461 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     2521 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     2581 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     2641 tgcggcgacc gagttgctct tgcccggcgt caacacggga taataccgcg ccacatagca
     2701 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     2761 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     2821 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     2881 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     2941 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     3001 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     3061 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtcttc
     3121 aagaattctc atgtttgaca gcttatcatc gataagcttt aatgcggtag tttatcacag
     3181 ttaaattgct aacgcagtca ggcaccgtgt atgaaatcta acaatgcgct catcgtcatc
     3241 ctcggcaccg tcaccctgga tgctgtaggc ataggcttgg ttatgccggt actgccgggc
     3301 ctcttgcggg atatcgtcca ttccgacagc atcgccagtc actatggcgt gctgctagcg
     3361 ctatatgcgt tgatgcaatt tctatgcgca cccgttctcg gagcactgtc cgaccgcttt
     3421 ggccgccgcc cagtcctgct cgcttcgcta cttggagcca ctatcgacta cgcgatcatg
     3481 gcgaccacac ccgtcctgtg gatcctctac gccggacgca tcgtggccgg catcaccggc
     3541 gccacaggtg cggttgctgg cgcctatatc gccgacatca ccgatgggga agatcgggct
     3601 cgccacttcg ggctcatgag cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc
     3661 gggggactgt tgggcgccat ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac
     3721 ggcctcaacc tactactggg ctgcttccta atgcaggagt cgcataaggg agagcgtcga
     3781 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     3841 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     3901 gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
     3961 tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
     4021 accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggccga cgcgctgggc
     4081 tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct tccccattat gattcttctc
     4141 gcttccggcg gcatcgggat gcccgcgttg caggccatgc tgtccaggca ggtagatgac
     4201 gaccatcagg gacagcttca aggatcgctc gcggctctta ccagcctaac ttcgatcatt
     4261 ggaccgctga tcgtcacggc gatttatgcc gcctcggcga gcacatggaa cgggttggca
     4321 tggattgtag gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg
     4381 agccgggcca cctcgacctg aatggaagcc ggcggcacct cgctaacgga ttcaccactc
     4441 caagaattgg agccaatcaa ttcttgcgga gaactgtgaa tgcgcaaacc aacccttggc
     4501 agaacatatc catcgcgtcc gccatctcca gcagccgcac gcggcgcatc tcgggcagcg
     4561 ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct
     4621 ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg
     4681 actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg
     4741 tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca
     4801 tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc
     4861 attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct
     4921 cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct
     4981 ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag
     5041 tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa
     5101 cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc
     5161 ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg
     5221 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     5281 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg caggtgtcgg ggcgcagcca
     5341 tgacccagtc accccatggt gcagtatgaa ggcggcggag ccgacaccac ggccaccgat
     5401 attatttgcc cgatgtacgc gcgcgtggat gaacaccagc ccttcccggc tttatcaaaa
     5461 agagtattga cttaaagtct aacctatagg atacttacag cgatggagag gtgtagtggt
     5521 aaccagaaga taagatggct ttcgctacct ggagagacgc gcccgctgat cctttgcgaa
     5581 tacgcccacg cgatgggtaa cagtcttggc ggtttcgcta aatactggca ggcgtttcgt
     5641 cagtatcccc gtttacaggg cggcttcgtc tgggactggg tggatcagtc gctgattaaa
     5701 tatgatgaaa acggcaaccc gtggtaccgg cttacggcgg tgattttggc gatacgccga
     5761 acgaattgtg agcggataac aattatcgcc agttctgtat gaacggtctg gtctttgccg
     5821 accgcacgcc gcatccagcg ctgacggaa
//
