LOCUS       sequence-341110-        3909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cell-free lead sensing reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3909)
  TITLE     Sensitizing cell-free molecular detection using cascaded genetic 
            circuitry
REFERENCE   2  (bases 1 to 3909)
  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"
COMMENT     Sequence Label: pJBL7053
FEATURES             Location/Qualifiers
     source          1..3909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(33..50)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(203..791)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(283..302)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(962..1753,1754..1822))
                     /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     1585..1604
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1823..1927)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1995..2013
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2051..2073)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2173..2192
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2386..2408
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2400..2417
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2401..2417
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(2548..2550,2551..3264)
                     /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             2548..3261
                     /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(2578..2606)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     3201..3221
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3399..3416)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3569..3615
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3711..3727)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3711..3727)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3724..3746)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3735..3751
                     /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(3759..3765,3766..3783,3784..3789))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3804..3825
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
       61 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
      121 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaggcc aggaaccgta
      181 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
      241 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
      301 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
      361 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
      421 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
      481 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
      541 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
      601 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
      661 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
      721 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
      781 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
      841 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
      901 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
      961 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     1021 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     1081 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     1141 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     1201 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     1261 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     1321 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     1381 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     1441 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     1501 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     1561 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     1621 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     1681 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     1741 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     1801 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     1861 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     1921 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     1981 cattaaccta taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg
     2041 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     2101 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     2161 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
     2221 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc
     2281 gcaactgttg ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag
     2341 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
     2401 gtaaaacgac ggccagtaag atctcggctg aattctaaaa gatctatgtc ttgactctat
     2461 agtaactaga gggtgttaaa tcggcaacgt cgactctcga gtgagattgt tgacggtacc
     2521 gtattttgga tctaggagga aggatctatg agcaaaggag aagaactttt cactggagtt
     2581 gtcccaattc ttgttgaatt agatggtgat gttaatgggc acaaattttc tgtccgtgga
     2641 gagggtgaag gtgatgctac aaacggaaaa ctcaccctta aatttatttg cactactgga
     2701 aaactacctg ttccgtggcc aacacttgtc actactctga cctatggtgt tcaatgcttt
     2761 tcccgttatc cggatcacat gaaacggcat gactttttca agagtgcaat gcccgaaggt
     2821 tatgtacagg aacgcactat atctttcaaa gatgacggga cctacaagac gcgtgctgaa
     2881 gtcaagtttg aaggtgatac ccttgttaat cgtatcgagt taaagggtat tgattttaaa
     2941 gaagatggaa acattcttgg acacaaactc gagtacaact ttaactcaca caatgtatac
     3001 atcacggcag acaaacaaaa gaatggaatc aaagctaact tcaaaattcg ccacaacgtt
     3061 gaagatggtt ccgttcaact agcagaccat tatcaacaaa atactccaat tggcgatggc
     3121 cctgtccttt taccagacaa ccattacctg tcgacacaat ctgtcctttc gaaagatccc
     3181 aacgaaaagc gtgaccacat ggtccttctt gagtttgtaa ctgctgctgg gattacacat
     3241 ggcatggatg agctctacaa ataaggatct gaagcttggg cccgaaaaaa aactcatctc
     3301 agaagaggat ctgaatagcg ccgtcgacca tcatcatcat cattgagttt aaacggtctc
     3361 cagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa
     3421 cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct
     3481 gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc
     3541 catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg
     3601 ggcctttcgt tttatctgtt gtttgtcggt gaactctgaa aggaggaact atatccggat
     3661 cgagatcctc tagagtcgac ctgcaggcat gcaagcttgg cgtaatcatg gtcatagctg
     3721 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     3781 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     3841 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3901 gcggggaga
//
