LOCUS       sequence-212218-        4977 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PTac driving GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4977)
  AUTHORS   Segall-Shapiro TH, Sontag ED, Voigt CA
  TITLE     Engineered promoters enable constant gene expression at any copy 
            number in bacteria.
  JOURNAL   Nat Biotechnol. 2018 Mar 19. pii: nbt.4111. doi: 10.1038/nbt.4111.
  PUBMED    29553576
REFERENCE   2  (bases 1 to 4977)
  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"; journalName: "Nat 
            Biotechnol. 2018 Mar 19. pii: nbt.4111. doi: 10.1038/nbt.4111."
COMMENT     Sequence Label: pTHSSe_37
FEATURES             Location/Qualifiers
     source          1..4977
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(3..8,9..24,25..31)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..56
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     misc_RNA        74..123
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(166..168,169..882)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     CDS             166..879
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Nager et al., 2011)"
                     /label=superfolder GFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(196..224)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     819..839
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      899..970
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      986..1013
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    1020..1040
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      1041..1098
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     CDS             complement(1256..2047)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPVESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRTDQLEEFVHYVKGEITKVVGK"
     rep_origin      complement(2642..3187)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2722..2741)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3655..3673
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      complement(3677..3720)
                     /label=bacterial terminator
                     /note="putative bacterial transcription terminator"
     CDS             complement(3763..4845)
                     /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     4807..4826
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc acaattctat
       61 ggactatgtt tagctgtcac cggatgtgct ttccggtctg atgagtccgt gaggacgaaa
      121 cagcctctac aaataatttt gtttaatcac acaggaaagt actagatgag caaaggagaa
      181 gaacttttca ctggagttgt cccaattctt gttgaattag atggtgatgt taatgggcac
      241 aaattttctg tccgtggaga gggtgaaggt gatgctacaa acggaaaact cacccttaaa
      301 tttatttgca ctactggaaa actacctgtt ccgtggccaa cacttgtcac tactctgacc
      361 tatggtgttc aatgcttttc ccgttatccg gatcacatga aacggcatga ctttttcaag
      421 agtgccatgc ccgaaggtta tgtacaggaa cgcactatat ctttcaaaga tgacgggacc
      481 tacaagacgc gtgctgaagt caagtttgaa ggtgataccc ttgttaatcg tatcgagtta
      541 aagggtattg attttaaaga agatggaaac attcttggac acaaactcga gtacaacttt
      601 aactcacaca atgtatacat cacggcagac aaacaaaaga atggaatcaa agctaacttc
      661 aaaattcgcc acaacgttga agatggttcc gttcaactag cagaccatta tcaacaaaat
      721 actccaattg gcgatggccc tgtcctttta ccagacaacc attacctgtc gacacaatct
      781 gtcctttcga aagatcccaa cgaaaagcgt gaccacatgg tccttcttga gtttgtaact
      841 gctgctggga ttacacatgg catggatgag ctctacaaat aatactagag ccaggcatca
      901 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
      961 gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct gcgtttatat
     1021 actagtagcg gccgctgcag tccggcaaaa aagggcaagg tgtcaccacc ctgccctttt
     1081 tctttaaaac cgaaaagatt acttcgcgtt atgcaggctt cctcgctcac tgactcgctg
     1141 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatctcgagt
     1201 cccgtcaagt cagcgtaatg ctctgccagt gttacaacca attaaccaat tctgattatt
     1261 tgccgactac cttggtgatc tcgcctttca cgtagtggac aaattcttcc aactgatctg
     1321 tgcgcgaggc caagcgatct tcttcttgtc caagataagc ctgtctagct tcaagtatga
     1381 cgggctgata ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg
     1441 cgattttgcc ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct
     1501 catcgccagc ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa
     1561 atagatcctg ttcaggaacc ggatcaaaga gttcctccgc cgctggacct accaaggcaa
     1621 cgctatgttc tcttgctttt gtcagcaaga tagccagatc aatgtcgatc gtggctggct
     1681 cgaagatacc tgcaagaatg tcattgcgct gccattctcc aaattgcagt tcgcgcttag
     1741 ctggataacg ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga
     1801 gaatctcgct ctctacaggg gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc
     1861 gcgttgtttc atcaagcctt acggtcaccg taaccagcaa atcaatatca ctgtgtggct
     1921 tcaggccgcc atccactgcg gagccgtaca aatgtacggc cagcaacgtc ggttcgagat
     1981 ggcgctcgat gacgccaact acctctgata gttgagtcga tacttcggcg atcaccgctt
     2041 ccctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg
     2101 atatattttt atcttgtgca atgtaacatc agagattttg agacacaacg tggctttgtt
     2161 gaataaatcg aacttttgct gagttgaagg atcagatcac gcatcttccc gacaacgcag
     2221 accgttccgt ggcaaagcaa aagttcaaaa tcaccaactg gtccacctac aacaaagctc
     2281 tcatcaaccg tggctccctc actttctggc tggatgatgg ggcgattcag gcctggtatg
     2341 agtcagcaac accttcttca cgaggcagac ctcagcgcta gcggagtgta tactggctta
     2401 ctatgttggc actgatgagg gtgtcagtga agtgcttcat gtggcaggag aaaaaaggct
     2461 gcaccggtgc gtcagcagaa tatgtgatac aggatatatt ccgcttcctc gctcactgac
     2521 tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg gggcggagat
     2581 ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg gcaaagccgt
     2641 ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca aatcagtggt
     2701 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggcggc tccctcgtgc
     2761 gctctcctgt tcctgccttt cggtttaccg gtgtcattcc gctgttatgg ccgcgtttgt
     2821 ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct ggactgtatg
     2881 cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2941 aacccggaaa gacatgcaaa agcaccactg gcagcagcca ctggtaattg atttagagga
     3001 gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt ttggtgactg
     3061 cgctcctcca agccagttac ctcggttcaa agagttggta gctcagagaa ccttcgaaaa
     3121 accgccctgc aaggcggttt tttcgttttc agagcaagag attacgcgca gaccaaaacg
     3181 atctcaagaa gatcatctta ttaaggggtc tgacgctcag tggaacgaaa actcacgtta
     3241 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3301 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     3361 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3421 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3481 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     3541 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattcc
     3601 atggtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     3661 cgtatcacga ggcagaattt cagataaaaa aaatccttag ctttcgctaa ggatgatttc
     3721 tggtcggcca attcgcgcta acttacatta attgcgttgc gctcactgcc cgctttccag
     3781 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3841 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagactggca acagctgatt
     3901 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     3961 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tatcttcggt
     4021 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     4081 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     4141 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     4201 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     4261 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     4321 gaccagatgc tccacgccca gtcgcgtacc gtcctcatgg gagaaaataa tactgttgat
     4381 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     4441 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     4501 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     4561 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     4621 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     4681 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     4741 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     4801 gacaccggca tactctgcga catcgtataa cgttactggt ttcatattca ccaccctgaa
     4861 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggc
     4921 gcgccgcttc gtcaggccac atagctttct tgttctgatc ggaacgatcg ttggctg
//
