LOCUS       sequence-400059-        7630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SR latch circuit on pLW555 backbone: PTac-S3, PTet-E1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7630)
  AUTHORS   Lebovich M, Zeng M, Andrews LB
  TITLE     Algorithmic Programming of Sequential Logic and Genetic Circuits for
            Recording Biochemical Concentration in a Probiotic Bacterium.
  JOURNAL   ACS Synth Biol. 2023 Aug 15. doi: 10.1021/acssynbio.3c00232.
  PUBMED    37581922
REFERENCE   2  (bases 1 to 7630)
  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: "ACS Synth 
            Biol. 2023 Aug 15. doi: 10.1021/acssynbio.3c00232."
COMMENT     Sequence Label: pML12
FEATURES             Location/Qualifiers
     source          1..7630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(399..1214)
                     /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(399..1214)
                     /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     1122..1141
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1206..1225
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        join(1393..1398,1399..1414,1415..1421)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1427..1446
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     protein_bind    2480..2498
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2505..2523
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        3289..3323
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     RBS             3332..3343
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4199..4210
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4328..5410
                     /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(4347..4366)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5429..6052
                     /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(5502..5521)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(7011..7030)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      complement(join(7395..7630,1..310))
                     /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(7475..7494)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 atgcacgaac cccccgttca gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag
       61 tccaacccgg aaagacatgc aaaagcacca ctggcagcag ccactggtaa ttgatttaga
      121 ggagttagtc ttgaagtcat gcgccggtta aggctaaact gaaaggacaa gttttggtga
      181 ctgcgctcct ccaagccagt tacctcggtt caaagagttg gtagctcaga gaaccttcga
      241 aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa
      301 acgatctcaa gaagatcatc ttattaaggg gtctgacgct cagtggaacg aaaaatcaat
      361 ctaaagtata tatgagtaaa cttggtctga cagttacctt agaaaaactc atcgagcatc
      421 aaatgaaact gcaatttatt catatcagga ttatcaatac catatttttg aaaaagccgt
      481 ttctgtaatg aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
      541 cggtctgcga ttccgactcg tccaacatca atacaaccta ttaatttccc ctcgtcaaaa
      601 ataaggttat caagtgagaa atcaccatga gtgacgactg aatccggtga gaatggcaaa
      661 agcttatgca tttctttcca gacttgttca acaggccagc cattacgctc gtcatcaaaa
      721 tcactcgcat caaccaaacc gttattcatt cgtgattgcg cctgagcgag acgaaatacg
      781 cgatcgctgt taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
      841 gccagcgcat caacaatatt ttcacctgaa tcaggatatt cttctaatac ctggaatgct
      901 gttttcccgg ggatcgcagt ggtgagtaac catgcatcat caggagtacg gataaaatgc
      961 ttgatggtcg gaagaggcat aaattccgtc agccagttta gtctgaccat ctcatctgta
     1021 acatcattgg caacgctacc tttgccatgt ttcagaaaca actctggcgc atcgggcttc
     1081 ccatacaatc gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
     1141 ccatataaat cagcatccat gttggaattt aatcgcggcc tggagcaaga cgtttcccgt
     1201 tgaatatggc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     1261 catgatgata tatttttatc ttgtgcaatg tacatcagag attttgagac acaaccaatt
     1321 attgaaggcc tccctaacgg ggggcctttt tttgtttctg gtctcccgct tggagaacga
     1381 tcgttggctg tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc
     1441 acaatttacg agcgcgggtg agagggattc gttaccaata gacaattgat tggacgttca
     1501 atataatgct agcctgagcg ctcaacgggt gtgcttcccg ttctgatgag tccgtgagga
     1561 cgaaagcgcc tctacaaata attttgttta agagtctatg gactatgttt tcacaaagga
     1621 agtaccagga tggcacgtaa aaccgcagca gaagcagaag aaacccgtca gcgtattatt
     1681 gatgcagcac tggaagtttt tgttgcacag ggtgttagtg atgcaaccct ggatcagatt
     1741 gcacgtaaag ccggtgttac ccgtggtgca gtttattggc attttaatgg taaactggaa
     1801 gttctgcagg cagttctggc aagccgtcag catccgctgg aactggattt tacaccggat
     1861 ctgggtattg aacgtagctg ggaagcagtt gttgttgcaa tgctggatgc agttcatagt
     1921 ccgcagagca aacagtttag cgaaattctg atttatcagg gtctggatga aagcggtctg
     1981 attcataatc gtatggttca ggcaagcgat cgttttctgc agtatattca tcaggttctg
     2041 cgtcatgcag ttacccaggg tgaactgccg attaatctgg atctgcagac cagcattggt
     2101 gtttttaaag gtctgattac cggtctgctg tatgaaggtc tgcgtagcaa agatcagcag
     2161 gcacagatta tcaaagttgc actgggtagc ttttgggcac tgctgcgtga accgcctcgt
     2221 tttctgctgt gtgaagaagc acagattaaa caggtgaaat ccttcgaata attcagccaa
     2281 aaaacttaag accgccggtc ttgtccacta ccttgcagta atgcggtgga caggatcggc
     2341 ggttttcttt tctcttctca aaggtcgctg gagtctatga ttggtccaga ttcgttacca
     2401 attgacagct agctcagtcc taggtatata catacatgct tgtttgtttg taaactacgt
     2461 actccaccgt tggctttttt ccctatcagt gatagagatt gacatcccta tcagtgatag
     2521 agataatgag cacctgagaa gtcaattaat gtgcttttaa ttctgatgag tcggtgacga
     2581 cgaaacttcc tctacaaata attttgttta accccccgag gagtagcaca tgccgaaact
     2641 gggtatgcag agcattcgtc gtcgtcagct gattgatgca accctggaag caattaatga
     2701 agttggtatg catgatgcaa ccattgcaca gattgcacgt cgtgccggtg ttagcaccgg
     2761 tattattagc cattatttcc gcgataaaaa cggtctgctg gaagcaacca tgcgtgatat
     2821 taccagccag ctgcgtgatg cagttctgaa tcgtctgcat gcactgccgc agggtagcgc
     2881 agaacagcgt ctgcaggcaa ttgttggtgg taattttgat gaaacccagg ttagcagcgc
     2941 agcaatgaaa gcatggctgg cattttgggc aagcagcatg catcagccga tgctgtatcg
     3001 tctgcagcag gttagcagtc gtcgtctgct gagcaatctg gttagcgaat ttcgtcgtga
     3061 actgcctcgt gaacaggcac aagaggcagg ttatggtctg gcagcactga ttgatggtct
     3121 gtggctgcgt gcagcactga gcggtaaacc gctggataaa acccgtgcaa atagcctgac
     3181 ccgtcatttt atcacccagc atctgccgac cgattaacca attattgaac acccttcggg
     3241 gtgttttttt gtttctggtc taccaggtaa tggtcaccat atatcaagtt tacggctagc
     3301 tcagtcctag gtactatgct agctactaga gaaagaggag aaatactaga tggctgaagc
     3361 gcaaaatgat cccctgctgc cgggatactc gtttaatgcc catctggtgg cgggtttaac
     3421 gccgattgag gccaacggtt atctcgattt ttttatcgac cgaccgctgg gaatgaaagg
     3481 ttatattctc aatctcacca ttcgcggtca gggggtggtg aaaaatcagg gacgagaatt
     3541 tgtttgccga ccgggtgata ttttgctgtt cccgccagga gagattcatc actacggtcg
     3601 tcatccggag gctcgcgaat ggtatcacca gtgggtttac tttcgtccgc gcgcctactg
     3661 gcatgaatgg cttaactggc cgtcaatatt tgccaatacg gggttctttc gcccggatga
     3721 agcgcaccag ccgcatttca gcgacctgtt tgggcaaatc attaacgccg ggcaagggga
     3781 agggcgctat tcggagctgc tggcgataaa tctgcttgag caattgttac tgcggcgcat
     3841 ggaagcgatt aacgagtcgc tccatccacc gatggataat cgggtacgcg aggcttgtca
     3901 gtacatcagc gatcacctgg cagacagcaa ttttgatatc gccagcgtcg cacagcatgt
     3961 ttgcttgtcg ccgtcgcgtc tgtcacatct tttccgccag cagttaggga ttagcgtctt
     4021 aagctggcgc gaggaccaac gtatcagcca ggcgaagctg cttttgagca ccacccggat
     4081 gcctatcgcc accgtcggtc gcaatgttgg ttttgacgat caactctatt tctcgcgggt
     4141 atttaaaaaa tgcaccgggg ccagcccgag cgagttccgt gccggttaat aaccaattat
     4201 tgaaggccgc taacgcagcc tttttttgtt tctggtctcc caatggcggc gcgccatcga
     4261 atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt
     4321 ggtgaatatg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca
     4381 gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt
     4441 ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg
     4501 caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca
     4561 aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat
     4621 ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg
     4681 cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc
     4741 tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag
     4801 tattattttc tcccatgagg acggtacgcg actgggcgtg gagcatctgg tcgcattggg
     4861 tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct
     4921 ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg
     4981 cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt
     5041 tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac
     5101 cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga
     5161 tagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca
     5221 aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct
     5281 gttgccagtc tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc
     5341 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
     5401 cgggcagtga caatccagga ggaaaaaaat gtccagatta gataaaagta aagtgattaa
     5461 cagcgcatta gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc gtaaactcgc
     5521 ccagaagcta ggtgtagagc agcctacatt gtattggcat gtaaaaaata agcgggcttt
     5581 gctcgacgcc ttagccattg agatgttaga taggcaccat actcactttt gccctttaga
     5641 aggggaaagc tggcaagatt ttttacgtaa taacgctaaa agttttagat gtgctttact
     5701 aagtcatcgc gatggagcaa aagtacattt aggtacacgg cctacagaaa aacagtatga
     5761 aactctcgaa aatcaattag cctttttatg ccaacaaggt ttttcactag agaatgcatt
     5821 atatgcactc agcgctgtgg ggcattttac tttaggttgc gtattggaag atcaagagca
     5881 tcaagtcgct aaagaagaaa gggaaacacc tactactgat agtatgccgc cattattacg
     5941 acaagctatc gaattatttg atcaccaagg tgcagagcca gccttcttat tcggccttga
     6001 attgatcata tgcggattag aaaaacaact taaatgtgaa agtgggtcct aaatcacaca
     6061 ggaaaggcct cgatgaaaaa cataaatgcc gacgacacat acagaataat taataaaatt
     6121 aaagcttgta gaagcaataa tgatattaat caatgcttat ctgatatgac taaaatggta
     6181 cattgtgaat attatttact cgcgatcatt tatcctcatt ctatggttaa atctgatatt
     6241 tcaatcctag ataattaccc taaaaaatgg aggcaatatt atgatgacgc taatttaata
     6301 aaatatgatc ctatagtaga ttattctaac tccaatcatt caccaattaa ttggaatata
     6361 tttgaaaaca atgctgtaaa taaaaaatct ccaaatgtaa ttaaagaagc gaaaacatca
     6421 ggtcttatca ctgggtttag tttccctatt catacggcta acaatggctt cggaatgctt
     6481 agttttgcac attcagaaaa agacaactat atagatagtt tatttttaca tgcgtgtatg
     6541 aacataccat taattgttcc ttctctagtt gataattatc gaaaaataaa tatagcaaat
     6601 aataaatcaa acaacgattt aaccaaaaga gaaaaagaat gtttagcgtg ggcatgcgaa
     6661 ggaaaaagct cttgggatat ttcaaaaata ttaggttgca gtgagcgtac tgtcactttc
     6721 catttaacca atgcgcaaat gaaactcaat acaacaaacc gctgccaaag tatttctaaa
     6781 gcaattttaa caggagcaat tgattgccca tactttaaaa attaaccata attggtaacg
     6841 aatcagacaa ttgacggctc gagggagtag catagggttt gcagaatccc tgcttcgtcc
     6901 atttgacagg cacattatgc atcgatgata agctgtcaaa catgagcaga tcctctacgc
     6961 cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc
     7021 cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagca aatattttat
     7081 ctgaggtgct tcctcgctca ctgactcgct gcacgaggca gacctcagcg ctagcggagt
     7141 gtatactggc ttactatgtt ggcactgatg agggtgtcag tgaagtgctt catgtggcag
     7201 gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga tacaggatat attccgcttc
     7261 ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa tggcttacga
     7321 acggggcgga gatttcccgg aagatgccag gaagatactt aacagggaag tgagagggcc
     7381 gcggcaaagc cgtttttcca taggctccgc ccccctgaca agcatcacga aatctgacgc
     7441 tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctggc
     7501 ggctccctcg tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat tccgctgtta
     7561 tggccgcgtt tgtctcattc cacgcctgac actcagttcc gggtaggcag ttcgctccaa
     7621 gctggactgt
//
