LOCUS       sequence-273107-        3905 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive sfGFP expression (high), bacterial promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3905)
  AUTHORS   Silverman AD, Kelley-Loughnane N, Lucks JB, Jewett MC
  TITLE     Deconstructing Cell-Free Extract Preparation for in Vitro Activation
            of Transcriptional Genetic Circuitry.
  JOURNAL   ACS Synth Biol. 2019 Feb 15;8(2):403-414. doi: 
            10.1021/acssynbio.8b00430. Epub 2019 Jan 29.
  PUBMED    30596483
REFERENCE   2  (bases 1 to 3905)
  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: 
            "10.1021/acssynbio.8b00430"; journalName: "ACS Synth Biol"; date: 
            "2019-02-15- 15"; volume: "8"; issue: "2"; pages: "403-414"
COMMENT     Sequence Label: pJBL7010
FEATURES             Location/Qualifiers
     source          1..3905
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        24..58
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     RBS             127..135
                     /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(141..143,144..857)
                     /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             141..854
                     /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(171..199)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     794..814
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             877..906
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(995..1012)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1165..1211
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(1307..1323)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1307..1323)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1320..1342)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1331..1347
                     /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(1355..1361,1362..1379,1380..1385))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1400..1421
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1538..1555)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1708..2296)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1788..1807)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2467..3258,3259..3327))
                     /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     3090..3109
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3328..3432)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3500..3518
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3556..3578)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3678..3697
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     join(3891..3905,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(3905,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtaga tctttgacag ctagctcagt cctaggtata atactagtac
       61 gtcgactctc gagtgagatt gttgacggta ccgtattttg gatctgagca aagcaataac
      121 agaacataag gaggtacgta atgagcaaag gagaagaact tttcactgga gttgtcccaa
      181 ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtccgt ggagagggtg
      241 aaggtgatgc tacaaacgga aaactcaccc ttaaatttat ttgcactact ggaaaactac
      301 ctgttccgtg gccaacactt gtcactactc tgacctatgg tgttcaatgc ttttcccgtt
      361 atccggatca catgaaacgg catgactttt tcaagagtgc aatgcccgaa ggttatgtac
      421 aggaacgcac tatatctttc aaagatgacg ggacctacaa gacgcgtgct gaagtcaagt
      481 ttgaaggtga tacccttgtt aatcgtatcg agttaaaggg tattgatttt aaagaagatg
      541 gaaacattct tggacacaaa ctcgagtaca actttaactc acacaatgta tacatcacgg
      601 cagacaaaca aaagaatgga atcaaagcta acttcaaaat tcgccacaac gttgaagatg
      661 gttccgttca actagcagac cattatcaac aaaatactcc aattggcgat ggccctgtcc
      721 ttttaccaga caaccattac ctgtcgacac aatctgtcct ttcgaaagat cccaacgaaa
      781 agcgtgacca catggtcctt cttgagtttg taactgctgc tgggattaca catggcatgg
      841 atgagctcta caaataagga tctgaagctt gggcccgaac aaaaactcat ctcagaagag
      901 gatctgaata gcgccgtcga ccatcatcat catcatcatt gagtttaaac ggtctccagc
      961 ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca
     1021 gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc
     1081 ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctccccatg
     1141 cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     1201 tttcgtttta tctgttgttt gtcggtgaac tctgaaagga ggaactatat ccggatcgag
     1261 atcctctaga gtcgacctgc aggcatgcaa gcttggcgta atcatggtca tagctgtttc
     1321 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     1381 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     1441 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     1501 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     1561 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1621 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aggccaggaa
     1681 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     1741 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     1801 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     1861 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     1921 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     1981 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     2041 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     2101 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     2161 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     2221 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     2281 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     2341 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     2401 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     2461 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     2521 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     2581 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     2641 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     2701 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     2761 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     2821 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     2881 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     2941 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     3001 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     3061 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     3121 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     3181 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     3241 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     3301 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     3361 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3421 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     3481 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     3541 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     3601 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     3661 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg
     3721 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag
     3781 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     3841 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     3901 acgtt
//
