LOCUS       sequence-405689-        5659 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  T7RNAP protein expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5659)
  AUTHORS   Koh HR, Roy R, Sorokina M, Tang GQ, Nandakumar D, Patel SS, Ha T
  TITLE     Correlating Transcription Initiation and Conformational Changes by a
            Single-Subunit RNA Polymerase with Near Base-Pair Resolution.
  JOURNAL   Mol Cell. 2018 May 17;70(4):695-706.e5. doi: 
            10.1016/j.molcel.2018.04.018.
  PUBMED    29775583
REFERENCE   2  (bases 1 to 5659)
  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: 
            "10.1016/j.molcel.2018.04"; journalName: "Mol Cell"; date: 
            "2018-05-17- 17"; volume: "70"; issue: "4"; pages: "695-706.e5"
COMMENT     Sequence Label: T7RNAP Cyslight V174C
FEATURES             Location/Qualifiers
     source          1..5659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     701..718
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    733..873
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(799..818)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     959..981
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     protein_bind    1066..1087
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1102..1107,1108..1125,1126..1132)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1140..1156
                     /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)."
     primer_bind     1145..1167
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1164..1180
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1164..1180
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     RBS             1229..1251
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1270..1287
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1297..1314
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     promoter        complement(join(4341..4346,4347..4363,4364..4369))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4396..4414)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4482..4586
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4587..4655,4656..5447)
                     /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     complement(4805..4824)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5618..5659,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga
      661 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga
      721 agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
      781 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
      841 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
      901 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
      961 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctggca
     1021 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     1081 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     1141 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc
     1201 atgcctgcag gtcgactcta gaaataattt tgtttaactt taagaaggag atataccatg
     1261 ggcagcagcc atcatcatca tcatcacagc agcggcctgg tgccgcgcgg cagccatatg
     1321 ctcgagatga acacgattaa catcgctaag aacgacttct ctgacatcga actggctgct
     1381 atcccgttca acactctggc tgaccattac ggtgagcgtt tagctcgcga acagttggcc
     1441 cttgagcatg agtcttacga gatgggtgaa gcacgcttcc gcaagatgtt tgagcgtcaa
     1501 cttaaagctg gtgaggttgc ggataacgct gccgccaagc ctctcatcac taccctactc
     1561 cctaagatga ttgcacgcat caacgactgg tttgaggaag tgaaagctaa gcgcggcaag
     1621 cgcccgacag ccttccagtt cctgcaagaa atcaagccgg aagccgtagc gtacatcacc
     1681 attaagacca ctctggctag cctaaccagt gctgacaata caaccgttca ggctgtagca
     1741 agcgcaatcg gtcgggccat tgaggacgag gctcgcttcg gtcgtatccg tgaccttgaa
     1801 gctaagcact tcaagaaaaa cgttgaggaa caactcaaca agcgctgcgg gcacgtctac
     1861 aagaaagcat ttatgcaagt tgtcgaggct gacatgctct ctaagggtct actcggtggc
     1921 gaggcgtggt cttcgtggca taaggaagac tctattcatg taggagtacg ctgcatcgag
     1981 atgctcattg agtcaaccgg aatggttagc ttacaccgcc aaaatgctgg cgtagtaggt
     2041 caagactctg agactatcga actcgcacct gaatacgctg aggctatcgc aacccgtgca
     2101 ggtgcgctgg ctggcatctc tccgatgttc caaccttgcg tagttcctcc taagccgtgg
     2161 actggcatta ctggtggtgg ctattgggct aacggtcgtc gtcctctggc gctggtgcgt
     2221 actcacagta agaaagcact gatgcgctac gaagacgttt acatgcctga ggtgtacaaa
     2281 gcgattaaca ttgcgcaaaa caccgcatgg aaaatcaaca agaaagtcct agcggtcgcc
     2341 aacgtaatca ccaagtggaa gcatagcccg gtcgaggaca tccctgcgat tgagcgtgaa
     2401 gaactcccga tgaaaccgga agacatcgac atgaatcctg aggctctcac cgcgtggaaa
     2461 cgtgctgccg ctgctgtgta ccgcaaggac aaggctcgca agtctcgccg tatcagcctt
     2521 gagttcatgc ttgagcaagc caataagttt gctaaccata aggccatctg gttcccttac
     2581 aacatggact ggcgcggtcg tgtttacgct gtgtcaatgt tcaacccgca aggtaacgat
     2641 atgaccaaag gactgcttac gctggcgaaa ggtaaaccaa tcggtaagga aggttactac
     2701 tggctgaaaa tccacggtgc aaactgtgcg ggtgtcgata aggttccgtt ccctgagcgc
     2761 atcaagttca ttgaggaaaa ccacgagaac atcatggctt gcgctaagtc tccactggag
     2821 aacacttggt gggctgagca agattctccg ttcagcttcc ttgcgttcag ctttgagtac
     2881 gctggggtac agcaccacgg cctgagctat aacagctccc ttccgctggc gtttgacggg
     2941 tcttgctctg gcatccagca cttctccgcg atgctccgag atgaggtagg tggtcgcgcg
     3001 gttaacttgc ttcctagtga aaccgttcag gacatctacg ggattgttgc taagaaagtc
     3061 aacgagattc tacaagcaga cgcaatcaat gggaccgata acgaagtagt taccgtgacc
     3121 gatgagaaca ctggtgaaat ctctgagaaa gtcaagctgg gcactaaggc actggctggt
     3181 caatggctgg cttacggtgt tactcgcagt gtgactaagc gttcagtcat gacgctggct
     3241 tacgggtcca aagagttcgg cttccgtcaa caagtgctgg aagataccat tcagccagct
     3301 attgattccg gcaagggtct gatgttcact cagccgaatc aggctgctgg atacatggct
     3361 aagctgattt gggaatctgt gagcgtgacg gtggtagctg cggttgaagc aatgaactgg
     3421 cttaagtctg ctgctaagct gctggctgct gaggtcaaag ataagaagac tggagagatt
     3481 cttcgcaagc gtagcgctgt gcattgggta actcctgatg gtttccctgt gtggcaggaa
     3541 tacaagaagc ctattcagac gcgcttgaac ctgatgttcc tcggtcagtt ccgcttacag
     3601 cctaccatta acaccaacaa agatagcgag attgatgcac acaaacagga gtctggtatc
     3661 gctcctaact ttgtacacag ccaagacggt agccaccttc gtaagactgt agtgtgggca
     3721 cacgagaagt acggaatcga atcttttgca ctgattcacg actccttcgg taccattccg
     3781 gctgacgctg cgaacctgtt caaagcagtg cgcgaaacta tggttgacac atatgagtct
     3841 agcgatgtac tggctgattt ctacgaccag ttcgctgacc agttgcacga gtctcaattg
     3901 gacaaaatgc cagcacttcc ggctaaaggt aacttgaacc tccgtgacat cttagagtcg
     3961 gacttcgcgt tcgcgtaacg ccaaatcaat acgactccgg atccacagga cgggtgtggt
     4021 cgccatgatc gcgtagtcga tagtggctcc aagtagcgaa gcgagcagga ctgggcggcg
     4081 gccaaagcgg tcggacagtg ctccgagaac gggtgcgcat agaaattgca tcaacgcata
     4141 tagcgctagc agcacgccat agtgactggc gatgctgtcg gaatggacga tatcccgcaa
     4201 gaggcccggc agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc
     4261 gaggatgacg atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt
     4321 taactgtgat aaactaccgc attaaagctt atcgatgata agctgtcaaa catgagaatt
     4381 cttgaagacg aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa
     4441 tggtttctta gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     4501 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     4561 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     4621 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     4681 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     4741 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     4801 ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc
     4861 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     4921 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     4981 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     5041 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     5101 caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat
     5161 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     5221 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     5281 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     5341 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     5401 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     5461 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     5521 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     5581 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     5641 taatctgctg cttgcaaac
//
