LOCUS       sequence-378045-        6655 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gamma-aminobutyric acid sensor expressing eYFP reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6655)
  AUTHORS   Lebovich M, Andrews LB
  TITLE     Surveying the Genetic Design Space for Transcription Factor-Based 
            Metabolite Biosensors: Synthetic Gamma-Aminobutyric Acid and 
            Propionate Biosensors in E. coli Nissle 1917.
  JOURNAL   Front Bioeng Biotechnol. 2022 Aug 25;10:938056. doi: 
            10.3389/fbioe.2022.938056. eCollection 2022.
  PUBMED    36091463
REFERENCE   2  (bases 1 to 6655)
  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: "2022-08-25"; volume: "10:938056. doi"; 
            pages: " 10.3389/fbioe.2022.938056. eCollection 2022"
COMMENT     Sequence Label: pML3009
FEATURES             Location/Qualifiers
     source          1..6655
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        29..63
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
     CDS             1644..1655
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(1863..1880)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1997..2542)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(2077..2096)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2631..3446)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2631..3446)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3354..3373
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3438..3457
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     misc_RNA        3682..3731
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(3781..3783,3784..3786,3787..4500)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3826..3847)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4087..4106)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4434..4455
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4660..5742
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4679..4698)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5761..6384
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(5834..5853)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(6567..6586)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 acacagagca gagaggacga tactggagtt tacggctagc tcagtcctag gtatagtgct
       61 agcctgagtc cgagtgctta cagttctgaa ggagtcaatt aatgtgcttt taattctgat
      121 gagacggtga cgtcgaaact ccctctacaa ataattttgt ttaataacat gttgtcaatg
      181 aggagctatc ttgatggata tcacgattac actcgatcgt tcagaacaag ccgattatat
      241 ctatcagcaa atttatcaaa agctgaaaaa agaaatcctc agccgcaatc tgctgccgca
      301 ctcgaaggtt ccctccaagc gggagctggc tgaaaatctc aaggtcagcg taaattcagt
      361 gaattcagcc tatcagcagc tgctggctga ggggtatttg tacgccattg aacgaaaggg
      421 tttcttcgtg gaggaactag acatgttttc cgccgaggag caccctccat ttgcactgcc
      481 ggatgaccta aaagagattc acatcgacca gagcgattgg atatcgtttt cacacatgag
      541 ttccgataca gaccattttc cgatcaaaag ctggttccgc tgcgagcaaa aagcggcctc
      601 ccgctcatac cgcacgctcg gcgatatgtc acatccgcaa gggatatatg aagtgagagc
      661 ggccattacg aggctcattt ccctgacgag gggtgtaaaa tgcaggccgg aacaaatgat
      721 cataggggca ggcacacagg tgctcatgca gctgttgact gagcttttac ccaaggaagc
      781 cgtgtatgcg atggaggagc ctggctacag gcgcatgtat cagcttttga agaatgccgg
      841 aaaacaagta aagacgatca tgctggatga aaaaggcatg tcgattgctg aaatcaccag
      901 acagcagcca gatgtgctgg tgaccacccc gtcgcatcag tttccgtccg gaacgattat
      961 gcctgtatcc agaagaattc agctgctgaa ctgggcagcc gaggagccgc gccgatatat
     1021 cattgaggac gattatgata gtgaattcac atatgatgta gacagtattc cggcgctgca
     1081 aagcctcgac cgttttcaaa atgtcatcta tatgggaacc ttttcaaagt cccttctccc
     1141 cggcttacgg atcagctata tggtgttgcc gcctgagctg ttgagggcat acaaacagcg
     1201 gggctatgat ctgcagactt gctcatcact cacacagctc accctgcagg aatttatcga
     1261 gtctggtgaa tatcagaagc atataaaaaa aatgaagcag cattataaag aaaagagaga
     1321 acgcctgatc accgctttag aagcagagtt cagcggagag gttaccgtaa aaggggcaaa
     1381 tgcggggctg cattttgtta ccgaatttga taccaggcgc accgaacaag acatcctgtc
     1441 acatgctgcc gggctgcagc ttgaaatatt cggaatgagc cgatttaact tgaaggaaaa
     1501 caagcggcaa acgggcaggc ctgctctcat tatcggcttt gcacggctga aggaagaaga
     1561 tattcaggag ggtgtgcagc ggcttttcaa agcggtttac ggacataaaa aaatccccgt
     1621 tacaggggat tgactgacca attattgaag gccgctaacg cggccttttt ttgtttctgg
     1681 tcacccatcg gcaggttgcc aggtctccag gcagaatcgg cagacctcag cgctagcgga
     1741 gtgtatactg gcttactatg ttggcactga tgagggtgtc agtgaagtgc ttcatgtggc
     1801 aggagaaaaa aggctgcacc ggtgcgtcag cagaatatgt gatacaggat atattccgct
     1861 tcctcgctca ctgactcgct acgctcggtc gttcgactgc ggcgagcgga aatggcttac
     1921 gaacggggcg gagatttccc ggaagatgcc aggaagatac ttaacaggga agtgagaggg
     1981 ccgcggcaaa gccgtttttc cataggctcc gcccccctga caagcatcac gaaatctgac
     2041 gctcaaatca gtggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     2101 gcggctccct cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt
     2161 tatggccgcg tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc
     2221 aagctggact gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac
     2281 tatcgtcttg agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt
     2341 aattgattta gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac
     2401 aagttttggt gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca
     2461 gagaaccttc gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac
     2521 gcgcagacca aaacgatctc aagaagatca tcttattaag gggtctgacg ctcagtggaa
     2581 cgaaaaatca atctaaagta tatatgagta aacttggtct gacagttacc ttagaaaaac
     2641 tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     2701 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     2761 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     2821 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     2881 gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc
     2941 tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     3001 agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg
     3061 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     3121 acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta
     3181 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc
     3241 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     3301 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     3361 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctggagcaa
     3421 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     3481 agttttattg ttcatgatga tatattttta tcttgtgcaa tgtacatcag agattttgag
     3541 acacaaccaa ttattgaagg cctccctaac ggggggcctt tttttgtttc tggtctcccg
     3601 cttggagtcc tgataccatc aaaaagttat aattggtact ttacggcata ccaagtccta
     3661 ggtactatgc tagcgctgaa gctgtcaccg gatgtgcttt ccggtctgat gagtccgtga
     3721 ggacgaaaca gcctctacaa ataattttgt ttaatactag agaaagaggg gaaatactag
     3781 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
     3841 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac
     3901 ggcaagctga ccctgaagtt catctgcacc acaggcaagc tgcccgtgcc ctggcccacc
     3961 ctcgtgacca ccttcggcta cggcctgcaa tgcttcgccc gctaccccga ccacatgaag
     4021 ctgcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     4081 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     4141 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     4201 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     4261 ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     4321 gaccactacc agcagaacac cccaatcggc gacggccccg tgctgctgcc cgacaaccac
     4381 taccttagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     4441 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     4501 ctcggtacca aattccagaa aagaggcctc ccgaaagggg ggcctttttt cgttttggtc
     4561 caatgaggta atgaatggcg gcgcgccatc gaatggcgca aaacctttcg cggtatggca
     4621 tgatagcgcc cggaagagag tcaattcagg gtggtgaata tgaaaccagt aacgttatac
     4681 gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc
     4741 agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac
     4801 attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc
     4861 acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc
     4921 gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt
     4981 aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg
     5041 ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt
     5101 cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga ggacggtacg
     5161 cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc
     5221 ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc
     5281 aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa
     5341 caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat
     5401 cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat
     5461 atctcggtag tgggatacga cgataccgaa gatagctcat gttatatccc gccgttaacc
     5521 accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc
     5581 tctcagggcc aggcggtgaa gggcaatcag ctgttgccag tctcactggt gaaaagaaaa
     5641 accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     5701 cagctggcac gacaggtttc ccgactggaa agcgggcagt gacaatccag gaggaaaaaa
     5761 atgtccagat tagataaaag taaagtgatt aacagcgcat tagagctgct taatgaggtc
     5821 ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga gcagcctaca
     5881 ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat tgagatgtta
     5941 gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga ttttttacgt
     6001 aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc aaaagtacat
     6061 ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt agccttttta
     6121 tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt ggggcatttt
     6181 actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga aagggaaaca
     6241 cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt tgatcaccaa
     6301 ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt agaaaaacaa
     6361 cttaaatgtg aaagtgggtc ctaataattg gtaacgaatc agacaattga cggctcgagg
     6421 gagtagcata gggtttgcag aatccctgct tcgtccattt gacaggcaca ttatgcatcg
     6481 atgataagct gtcaaacatg agcagatcct ctacgccgga cgcatcgtgg ccggcatcac
     6541 cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg
     6601 ggctcgccac ttcgggctca tgagcaaata ttttatctga ggtgcttcct cgctc
//
