LOCUS       sequence-383080-        3520 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of superfolder GFP (sfGFP) by a hybrid T7 promoter with 
            tetO..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3520)
  AUTHORS   Deng Y, Beahm DR, Ran X, Riley TG, Sarpeshkar R
  TITLE     Rapid modeling of experimental molecular kinetics with simple 
            electronic circuits instead of with complex differential equations.
  JOURNAL   Front Bioeng Biotechnol. 2022 Sep 28;10:947508. doi: 
            10.3389/fbioe.2022.947508. eCollection 2022.
  PUBMED    36246369
REFERENCE   2  (bases 1 to 3520)
  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: "Front 
            Bioeng Biotechnol."; date: "2022-09-28"; volume: "10:947508. doi"; 
            pages: " 10.3389/fbioe.2022.947508. eCollection 2022"
COMMENT     Sequence Label: pDA313
FEATURES             Location/Qualifiers
     source          1..3520
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     88..105
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        190..208
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    212..230
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     RBS             286..294
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             join(300..302,303..1016)
                     /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             300..1013
                     /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(330..358)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     953..973
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(1091..1109)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1105..1152
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1249..1704
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1336..1355)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1546..1567
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1730..1834
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1835..1903,1904..2695)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2053..2072)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2866..3454
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3355..3374
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
       61 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
      121 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag gatctcgatc
      181 ccgcgaaatt aatacgactc actataggag gtccctatca gtgatagaga acgtcgactc
      241 tcgagtgaga ttgttgacgg taccgtattt tacgggaaga ttacataagg aggtttgtaa
      301 tgagcaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg
      361 atgttaatgg gcacaaattt tctgtccgtg gagagggtga aggtgatgct acaaacggaa
      421 aactcaccct taaatttatt tgcactactg gaaaactacc tgttccgtgg ccaacacttg
      481 tcactactct gacctatggt gttcaatgct tttcccgtta tccggatcac atgaaacggc
      541 atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaacgcact atatctttca
      601 aagatgacgg gacctacaag acgcgtgctg aagtcaagtt tgaaggtgat acccttgtta
      661 atcgtatcga gttaaagggt attgatttta aagaagatgg aaacattctt ggacacaaac
      721 tcgagtacaa ctttaactca cacaatgtat acatcacggc agacaaacaa aagaatggaa
      781 tcaaagctaa cttcaaaatt cgccacaacg ttgaagatgg ttccgttcaa ctagcagacc
      841 attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac aaccattacc
      901 tgtcgacaca atctgtcctt tcgaaagatc ccaacgaaaa gcgtgaccac atggtccttc
      961 ttgagtttgt aactgctgct gggattacac atggcatgga tgagctctac aaataaaggt
     1021 aagatctaga attcgaagct tgatccggct gctaacaaag cccgaaagga agctgagttg
     1081 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1141 aggggttttt tgctgaaagg aggaactata tccggatctg gcgtaatagc gaagaggccc
     1201 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
     1261 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1321 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1381 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1441 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1501 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1561 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1621 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1681 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1741 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     1801 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     1861 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     1921 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     1981 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2041 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     2101 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2161 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2221 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2281 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2341 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     2401 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2461 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2521 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2581 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2641 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2701 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     2761 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     2821 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     2881 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     2941 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3001 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3061 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3121 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3181 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3241 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3301 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3361 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3421 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3481 gttcctggcc ttttgctggc cttttgctca catgttcttt
//
