LOCUS       sequence-331128-        5956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cell-free cascaded nitrate sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5956)
  TITLE     Sensitizing cell-free molecular detection using cascaded genetic 
            circuitry
REFERENCE   2  (bases 1 to 5956)
  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: pJBL7065
FEATURES             Location/Qualifiers
     source          1..5956
                     /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             859..876
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3554..3583
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3599..3616
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3672..3689)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3842..3888
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(3984..4000)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3984..4000)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3997..4019)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4008..4024
                     /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(4032..4038,4039..4056,4057..4062))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4077..4098
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4215..4232)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4385..4973)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4465..4484)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5144..5935,5936..5956,1..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     5767..5786
                     /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 gaggatcgca tcaccatcac catcacggat ccaacacgat taacatcgct
      901 aagaacgact tctctgacat cgaactggct gctatcccgt tcaacactct ggctgaccat
      961 tacggtgagc gtttagctcg cgaacagttg gcccttgagc atgagtctta cgagatgggt
     1021 gaagcacgct tccgcaagat gtttgagcgt caacttaaag ctggtgaggt tgcggataac
     1081 gctgccgcca agcctctcat cactacccta ctccctaaga tgattgcacg catcaacgac
     1141 tggtttgagg aagtgaaagc taagcgcggc aagcgcccga cagccttcca gttcctgcaa
     1201 gaaatcaagc cggaagccgt agcgtacatc accattaaga ccactctggc ttgcctaacc
     1261 agtgctgaca atacaaccgt tcaggctgta gcaagcgcaa tcggtcgggc cattgaggac
     1321 gaggctcgct tcggtcgtat ccgtgacctt gaagctaagc acttcaagaa aaacgttgag
     1381 gaacaactca acaagcgcgt agggcacgtc tacaagaaag catttatgca agttgtcgag
     1441 gctgacatgc tctctaaggg tctactcggt ggcgaggcgt ggtcttcgtg gcataaggaa
     1501 gactctattc atgtaggagt acgctgcatc gagatgctca ttgagtcaac cggaatggtt
     1561 agcttacacc gccaaaatgc tggcgtagta ggtcaagact ctgagactat cgaactcgca
     1621 cctgaatacg ctgaggctat cgcaacccgt gcaggtgcgc tggctggcat ctctccgatg
     1681 ttccaacctt gcgtagttcc tcctaagccg tggactggca ttactggtgg tggctattgg
     1741 gctaacggtc gtcgtcctct ggcgctggtg cgtactcaca gtaagaaagc actgatgcgc
     1801 tacgaagacg tttacatgcc tgaggtgtac aaagcgatta acattgcgca aaacaccgca
     1861 tggaaaatca acaagaaagt cctagcggtc gccaacgtaa tcaccaagtg gaagcattgt
     1921 ccggtcgagg acatccctgc gattgagcgt gaagaactcc cgatgaaacc ggaagacatc
     1981 gacatgaatc ctgaggctct caccgcgtgg aaacgtgctg ccgctgctgt gtaccgcaag
     2041 gacaaggctc gcaagtctcg ccgtatcagc cttgagttca tgcttgagca agccaataag
     2101 tttgctaacc ataaggccat ctggttccct tacaacatgg actggcgcgg tcgtgtttac
     2161 gctgtgtcaa tgttcaaccc gcaaggtaac gatatgacca aaggactgct tacgctggcg
     2221 aaaggtaaac caatcggtaa ggaaggttac tactggctga aaatccacgg tgcaaactgt
     2281 gcgggtgtcg ataaggttcc gttccctgag cgcatcaagt tcattgagga aaaccacgag
     2341 aacatcatgg cttgcgctaa gtctccactg gagaacactt ggtgggctga gcaagattct
     2401 ccgttctgct tccttgcatt ctgctttgag tacgctgggg tacagcacca cggcctgagc
     2461 tataactgct cccttccgct ggcgtttgac gggtcttgct ctggcatcca gcacttctcc
     2521 gcgatgctcc gagatgaggt aggtggtcgc gcggttaact tgcttcctag tgaaaccgtt
     2581 caggacatct acgggattgt tgctaagaaa gtcaacgaga ttctacaagc agacgcaatc
     2641 aatgggaccg ataacgaagt agttaccgtg accgatgaga acactggtga aatctctgag
     2701 aaagtcaagc tgggcactaa ggcactggct ggtcaatggc tggcttacgg tgttactcgc
     2761 agtgtgacta agcgttcagt catgacgctg gcttacgggt ccaaagagtt cggcttccgt
     2821 caacaagtgc tggaagatac cattcagcca gctattgatt ccggcaaggg tctgatgttc
     2881 actcagccga atcaggctgc tggatacatg gctaagctga tttgggaatc tgtgagcgtg
     2941 acggtggtag ctgcggttga agcaatgaac tggcttaagt ctgctgctaa gctgctggct
     3001 gctgaggtca aagataagaa gactggagag attcttcgca agcgttgcgc tgcgcattgg
     3061 gtaactcctg atggtttccc tgtgtggcag gaatacaaga agcctattaa aacgcgcatc
     3121 tctataatgt tcctcggtca gttcacctta aagcctacca ttaacaccaa caaagatagc
     3181 gagattgatg cacgcaaaca ggtgtctggt atcgctccta actttgtaca cagccaagac
     3241 ggtagccacc ttcgtaagac tgtagtgtgg gcacacgaga agtacggaat cgaatctttt
     3301 gcactgattc acgactcctt cggtaccatt ccggctgacg ctgcgaacct gttcaaagca
     3361 gtgcgcgaaa ctatggttga cacatatgag tcttgtgatg tactggctga tttctacgac
     3421 cagttcgctg accagttgca cgagtctcaa ttggacaaaa tgccagcact tccggctaaa
     3481 ggtaacttga acctccgtga catcttagag tcggacttcg cgttcgcgta ataaggatct
     3541 gaagcttggg cacgaacaaa aactcatctc agaagaggat ctgaatagcg ccgtcgacca
     3601 tcatcatcat catcattgag tttaaacggt ctccagcttg gctgttttgg cggatgagag
     3661 aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat
     3721 ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa
     3781 cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca
     3841 tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc
     3901 ggtgaactct gaaaggagga actatatccg gatcgagatc ctctagagtc gacctgcagg
     3961 catgcaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     4021 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     4081 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4141 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4201 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4261 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4321 aagaacatgt gagcaaaagg ccagcaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4381 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4441 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4501 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4561 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4621 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4681 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4741 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4801 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4861 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4921 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4981 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5041 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5101 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5161 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5221 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5281 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5341 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5401 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5461 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5521 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5581 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5641 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5701 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5761 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5821 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5881 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5941 aaacaggaag gcaaaa
//
