LOCUS       sequence-331012-        3985 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cell-free nitrate sensing reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3985)
  TITLE     Sensitizing cell-free molecular detection using cascaded genetic 
            circuitry
REFERENCE   2  (bases 1 to 3985)
  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: pJBL7048
FEATURES             Location/Qualifiers
     source          1..3985
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(49..153)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     221..239
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(277..299)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     399..418
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     612..634
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     626..643
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     627..643
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             847..1560
                     /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
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITTDKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(877..905)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1500..1520
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     CDS             1583..1612
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             1628..1645
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1701..1718)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1871..1917
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2013..2029)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2013..2029)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2026..2048)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2037..2053
                     /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(2061..2067,2068..2085,2086..2091))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2106..2127
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2244..2261)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2414..3002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2494..2513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3173..3964,3965..3985,1..48))
                     /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     3796..3815
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
       61 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
      121 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
      181 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
      241 ctttcgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
      301 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
      361 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
      421 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
      481 caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
      541 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
      601 gccagggttt tcccagtcac gacgttgtaa aacgacggcc agtaagatct cggctgaatt
      661 ctaaagatct actgcatatt tgaaaattgc ccaaacgtac atgcccgaat gtacgttttt
      721 ttcatttcat tgtcaactac aatgagaaag aatgtgatca agcaatgtgt tgaaaggaga
      781 ttatcacgtc gactctcgag tgagattgtt gacggtaccg tattttggat ctaggaggaa
      841 ggatctatga gcaaaggaga agaacttttc actggagttg tcccaattct tgttgaatta
      901 gatggtgatg ttaatgggca caaattttct gtccgtggag agggtgaagg tgatgctaca
      961 aacggaaaac tcacccttaa atttatttgc actactggaa aactacctgt tccgtggcca
     1021 acacttgtca ctactctgac ctatggtgtt caatgctttt cccgttatcc ggatcacatg
     1081 aaacggcatg actttttcaa gagtgccatg cccgaaggtt atgtacagga acgcactata
     1141 tctttcaaag atgacgggac ctacaagacg cgtgctgaag tcaagtttga aggtgatacc
     1201 cttgttaatc gtatcgagtt aaagggtatt gattttaaag aagatggaaa cattcttgga
     1261 cacaaactcg agtacaactt taactcacac aatgtataca tcacgacaga caaacaaaag
     1321 aatggaatca aagctaactt caaaattcgc cacaacgttg aagatggttc cgttcaacta
     1381 gcagaccatt atcaacaaaa tactccaatt ggcgatggcc ctgtcctttt accagacaac
     1441 cattacctgt cgacacaatc tgtcctttcg aaagatccca acgaaaagcg tgaccacatg
     1501 gtccttcttg agtttgtaac tgctgctggg attacacatg gcatggatga gctctacaaa
     1561 taaggatctg aagcttgggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc
     1621 cgtcgaccat catcatcatc atcattgagt ttaaacggtc tccagcttgg ctgttttggc
     1681 ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag cggtctgata
     1741 aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca
     1801 gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac
     1861 tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     1921 ttgtttgtcg gtgaactctg aaaggaggaa ctatatccgg atcgagatcc tctagagtcg
     1981 acctgcaggc atgcaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     2041 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     2101 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2161 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2221 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     2281 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     2341 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaggc caggaaccgt aaaaaggccg
     2401 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     2461 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     2521 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     2581 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     2641 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     2701 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     2761 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     2821 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     2881 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     2941 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3001 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3061 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3121 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3181 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3241 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3301 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3361 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3421 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     3481 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     3541 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     3601 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     3661 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     3721 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     3781 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     3841 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     3901 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     3961 ggtgagcaaa aacaggaagg caaaa
//
