LOCUS       sequence-409401-        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
FEATURES             Location/Qualifiers
     source          1..5659
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(213..1004,1005..1073))
                     /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     836..855
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1074..1178)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1246..1264
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(1291..1296,1297..1313,1314..1319)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             complement(4346..4363)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(4373..4390)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4409..4431
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     primer_bind     complement(4480..4496)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4480..4496)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4493..4515)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4504..4520
                     /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)."
     promoter        complement(join(4528..4534,4535..4552,4553..4558))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4573..4594
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4679..4701)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4787..4927
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4842..4861
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4942..4959)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5113..5659,1..42))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5193..5212)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
       61 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
      121 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
      181 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
      241 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
      301 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
      361 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
      421 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
      481 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg
      541 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
      601 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
      661 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
      721 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
      781 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat
      841 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
      901 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
      961 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     1021 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     1081 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     1141 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     1201 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     1261 aggccctttc gtcttcaaga attctcatgt ttgacagctt atcatcgata agctttaatg
     1321 cggtagttta tcacagttaa attgctaacg cagtcaggca ccgtgtatga aatctaacaa
     1381 tgcgctcatc gtcatcctcg gcaccgtcac cctggatgct gtaggcatag gcttggttat
     1441 gccggtactg ccgggcctct tgcgggatat cgtccattcc gacagcatcg ccagtcacta
     1501 tggcgtgctg ctagcgctat atgcgttgat gcaatttcta tgcgcacccg ttctcggagc
     1561 actgtccgac cgctttggcc gccgcccagt cctgctcgct tcgctacttg gagccactat
     1621 cgactacgcg atcatggcga ccacacccgt cctgtggatc cggagtcgta ttgatttggc
     1681 gttacgcgaa cgcgaagtcc gactctaaga tgtcacggag gttcaagtta cctttagccg
     1741 gaagtgctgg cattttgtcc aattgagact cgtgcaactg gtcagcgaac tggtcgtaga
     1801 aatcagccag tacatcgcta gactcatatg tgtcaaccat agtttcgcgc actgctttga
     1861 acaggttcgc agcgtcagcc ggaatggtac cgaaggagtc gtgaatcagt gcaaaagatt
     1921 cgattccgta cttctcgtgt gcccacacta cagtcttacg aaggtggcta ccgtcttggc
     1981 tgtgtacaaa gttaggagcg ataccagact cctgtttgtg tgcatcaatc tcgctatctt
     2041 tgttggtgtt aatggtaggc tgtaagcgga actgaccgag gaacatcagg ttcaagcgcg
     2101 tctgaatagg cttcttgtat tcctgccaca cagggaaacc atcaggagtt acccaatgca
     2161 cagcgctacg cttgcgaaga atctctccag tcttcttatc tttgacctca gcagccagca
     2221 gcttagcagc agacttaagc cagttcattg cttcaaccgc agctaccacc gtcacgctca
     2281 cagattccca aatcagctta gccatgtatc cagcagcctg attcggctga gtgaacatca
     2341 gacccttgcc ggaatcaata gctggctgaa tggtatcttc cagcacttgt tgacggaagc
     2401 cgaactcttt ggacccgtaa gccagcgtca tgactgaacg cttagtcaca ctgcgagtaa
     2461 caccgtaagc cagccattga ccagccagtg ccttagtgcc cagcttgact ttctcagaga
     2521 tttcaccagt gttctcatcg gtcacggtaa ctacttcgtt atcggtccca ttgattgcgt
     2581 ctgcttgtag aatctcgttg actttcttag caacaatccc gtagatgtcc tgaacggttt
     2641 cactaggaag caagttaacc gcgcgaccac ctacctcatc tcggagcatc gcggagaagt
     2701 gctggatgcc agagcaagac ccgtcaaacg ccagcggaag ggagctgtta tagctcaggc
     2761 cgtggtgctg taccccagcg tactcaaagc tgaacgcaag gaagctgaac ggagaatctt
     2821 gctcagccca ccaagtgttc tccagtggag acttagcgca agccatgatg ttctcgtggt
     2881 tttcctcaat gaacttgatg cgctcaggga acggaacctt atcgacaccc gcacagtttg
     2941 caccgtggat tttcagccag tagtaacctt ccttaccgat tggtttacct ttcgccagcg
     3001 taagcagtcc tttggtcata tcgttacctt gcgggttgaa cattgacaca gcgtaaacac
     3061 gaccgcgcca gtccatgttg taagggaacc agatggcctt atggttagca aacttattgg
     3121 cttgctcaag catgaactca aggctgatac ggcgagactt gcgagccttg tccttgcggt
     3181 acacagcagc ggcagcacgt ttccacgcgg tgagagcctc aggattcatg tcgatgtctt
     3241 ccggtttcat cgggagttct tcacgctcaa tcgcagggat gtcctcgacc gggctatgct
     3301 tccacttggt gattacgttg gcgaccgcta ggactttctt gttgattttc catgcggtgt
     3361 tttgcgcaat gttaatcgct ttgtacacct caggcatgta aacgtcttcg tagcgcatca
     3421 gtgctttctt actgtgagta cgcaccagcg ccagaggacg acgaccgtta gcccaatagc
     3481 caccaccagt aatgccagtc cacggcttag gaggaactac gcaaggttgg aacatcggag
     3541 agatgccagc cagcgcacct gcacgggttg cgatagcctc agcgtattca ggtgcgagtt
     3601 cgatagtctc agagtcttga cctactacgc cagcattttg gcggtgtaag ctaaccattc
     3661 cggttgactc aatgagcatc tcgatgcagc gtactcctac atgaatagag tcttccttat
     3721 gccacgaaga ccacgcctcg ccaccgagta gacccttaga gagcatgtca gcctcgacaa
     3781 cttgcataaa tgctttcttg tagacgtgcc ctacgcgctt gttgagttgt tcctcaacgt
     3841 ttttcttgaa gtgcttagct tcaaggtcac ggatacgacc gaagcgagcc tcgtcctcaa
     3901 tggcccgacc gattgcgctt gctacagcct gaacggttgt attgtcagca ctggttaggc
     3961 tagccagagt ggtcttaatg gtgatgtacg ctacggcttc cggcttgatt tcttgcagga
     4021 actggaaggc tgtcgggcgc ttgccgcgct tagctttcac ttcctcaaac cagtcgttga
     4081 tgcgtgcaat catcttaggg agtagggtag tgatgagagg cttggcggca gcgttatccg
     4141 caacctcacc agctttaagt tgacgctcaa acatcttgcg gaagcgtgct tcacccatct
     4201 cgtaagactc atgctcaagg gccaactgtt cgcgagctaa acgctcaccg taatggtcag
     4261 ccagagtgtt gaacgggata gcagccagtt cgatgtcaga gaagtcgttc ttagcgatgt
     4321 taatcgtgtt catctcgagc atatggctgc cgcgcggcac caggccgctg ctgtgatgat
     4381 gatgatgatg gctgctgccc atggtatatc tccttcttaa agttaaacaa aattatttct
     4441 agagtcgacc tgcaggcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg
     4501 aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc
     4561 ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt
     4621 ccagtcggga aacctgtcgt gccagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
     4681 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     4741 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     4801 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     4861 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     4921 catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4981 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     5041 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     5101 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     5161 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     5221 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     5281 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     5341 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     5401 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     5461 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     5521 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     5581 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     5641 tggtagcggt ggttttttt
//
