LOCUS       sequence-95207-e        4943 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PTac GFP expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4943)
  AUTHORS   Segall-Shapiro TH, Meyer AJ, Ellington AD, Sontag ED, Voigt CA
  TITLE     A 'resource allocator' for transcription based on a highly 
            fragmented T7 RNA polymerase.
  JOURNAL   Mol Syst Biol. 2014 Jul 30;10(7):742.
  PUBMED    25080493
REFERENCE   2  (bases 1 to 4943)
  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: "Mol Syst 
            Biol."; date: "2014-07-30"; volume: "10(7)"; pages: "742"
COMMENT     Sequence Label: pTHSSd_34
FEATURES             Location/Qualifiers
     source          1..4943
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..3,4..717)
                     /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             1..714
                     /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(31..59)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     654..674
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      761..792
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     rep_origin      1303..1847
                     /direction=RIGHT
                     /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     1748..1767
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1964..1981
                     /label=L4440
                     /note="L4440 vector, forward primer"
     CDS             2556..3347
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     terminator      complement(3474..3503)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     CDS             complement(3566..4648)
                     /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     4610..4629
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4783..4788,4789..4804,4805..4811)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    4817..4836
                     /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        4852..4901
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
ORIGIN
        1 atgagcaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt
       61 gatgttaatg ggcacaaatt ttctgtccgt ggagagggtg aaggtgatgc tacaaacgga
      121 aaactcaccc ttaaatttat ttgcactact ggaaaactac ctgttccgtg gccaacactt
      181 gtcactactc tgacctatgg tgttcaatgc ttttcccgtt atccggatca catgaaacgg
      241 catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc
      301 aaagatgacg ggacctacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt
      361 aatcgtatcg agttaaaggg tattgatttt aaagaagatg gaaacattct tggacacaaa
      421 ctcgagtaca actttaactc acacaatgta tacatcacgg cagacaaaca aaagaatgga
      481 atcaaagcta acttcaaaat tcgccacaac gttgaagatg gttccgttca actagcagac
      541 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac
      601 ctgtcgacac aatctgtcct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt
      661 cttgagtttg taactgctgc tgggattaca catggcatgg atgagctcta caaataacgc
      721 tgcaggagtc actaagggtt agttagttag attagcagaa agtcaaaagc ctccgaccgg
      781 aggcttttga ctaaaacttc ccttggggtt atcattgggg ctcactcaaa ggcggtaatc
      841 agataaaaaa aatccttagc tttcgctaag gatgatttct gctagagatg gaatagactg
      901 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
      961 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     1021 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     1081 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     1141 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     1201 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     1261 ttcgttccac tgagcgtcag accccttaat aagatgatct tcttgagatc gttttggtct
     1321 gcgcgtaatc tcttgctctg aaaacgaaaa aaccgccttg cagggcggtt tttcgaaggt
     1381 tctctgagct accaactctt tgaaccgagg taactggctt ggaggagcgc agtcaccaaa
     1441 acttgtcctt tcagtttagc cttaaccggc gcatgacttc aagactaact cctctaaatc
     1501 aattaccagt ggctgctgcc agtggtgctt ttgcatgtct ttccgggttg gactcaagac
     1561 gatagttacc ggataaggcg cagcggtcgg actgaacggg gggttcgtgc atacagtcca
     1621 gcttggagcg aactgcctac ccggaactga gtgtcaggcg tggaatgaga caaacgcggc
     1681 cataacagcg gaatgacacc ggtaaaccga aaggcaggaa caggagagcg cacgagggag
     1741 ccgccagggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac cactgatttg
     1801 agcgtcagat ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaac ggctttgccg
     1861 cggccctctc acttccctgt taagtatctt cctggcatct tccaggaaat ctccgccccg
     1921 ttcgtaagcc atttccgctc gccgcagtcg aacgaccgag cgtagcgagt cagtgagcga
     1981 ggaagcggaa tatatcctgt atcacatatt ctgctgacgc accggtgcag ccttttttct
     2041 cctgccacat gaagcacttc actgacaccc tcatcagtgc caacatagta agccagtata
     2101 cactccgcta gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca taccaggcct
     2161 gaatcgcccc atcatccagc cagaaagtga gggagccacg gttgatgaga gctttgttgt
     2221 aggtggacca gttggtgatt ttgaactttt gctttgccac ggaacggtct gcgttgtcgg
     2281 gaagatgcgt gatctgatcc ttcaactcag caaaagttcg atttattcaa caaagccacg
     2341 ttgtgtctca aaatctctga tgttacattg cacaagataa aaatatatca tcatgaacaa
     2401 taaaactgtc tgcttacata aacagtaata caaggggtgt ttactagagg ttgatcgggc
     2461 acgtaagagg ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg
     2521 agttatcgag attttcagga gctaaggaag ctaaaatgag ggaagcggtg atcgccgaag
     2581 tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc
     2641 tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg
     2701 atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg
     2761 accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca
     2821 ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat
     2881 ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca
     2941 ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag
     3001 cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa
     3061 ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta
     3121 cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg
     3181 ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctagac
     3241 aggcttatct tggacaagaa gaagatcgct tggcctcgcg cgcagatcag ttggaagaat
     3301 ttgtccacta cgtgaaaggc gagatcacca aggtagtcgg caaataatac tagctccggc
     3361 aaaaaaacgg gcaaggtgtc accaccctgc cctttttctt taaaaccgaa aagattactt
     3421 cgcgtttgcc acctgacgtc taagaaaagg aatattcagc aatttgcccg tgccgaagaa
     3481 aggcccaccc gtgaaggtga gccagtgagt tgattgctac gtaatgtcgg ccaattcgcg
     3541 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3601 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     3661 gggtggtttt tcttttcacc agtgagactg gcaacagctg attgcccttc accgcctggc
     3721 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     3781 tgatggtggt taacggcggg atataacatg agctatcttc ggtatcgtcg tatcccacta
     3841 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     3901 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     3961 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     4021 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     4081 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     4141 ccagtcgcgt accgtcctca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     4201 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     4261 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     4321 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     4381 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     4441 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     4501 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     4561 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     4621 cgacatcgta taacgttact ggtttcatat tcaccaccct gaattgactc tcttccgggc
     4681 gctatcatgc cataccgcga aaggttttgc gccattcgat ggcgcgccgc ttcgtcaggc
     4741 cacatagctt tcttgttctg atcggaacga tcgttggctg tgttgacaat taatcatcgg
     4801 ctcgtataat gtgtggaatt gtgagcgctc acaattctat ggactatgtt tctgtcaccg
     4861 gatgtgcttt ccggtctgat gagtccgtga ggacgaaaca gcctctacaa ataattttgt
     4921 ttaatcacac aggaaagtac tag
//
