LOCUS       sequence-400055-        7675 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SR latch circuit on pLW555 backbone: PTac-S2, PTet-H1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7675)
  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 7675)
  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: pML6
FEATURES             Location/Qualifiers
     source          1..7675
                     /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        3334..3368
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     RBS             3377..3388
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             4244..4255
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             4373..5455
                     /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(4392..4411)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             5474..6097
                     /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(5547..5566)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(7056..7075)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      complement(join(7440..7675,1..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(7520..7539)
                     /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 agcaggaatc tgaacgattc gttaccaatt gccatattta aaattcttgt
     1501 ttaaaatgct agcctgagcg ctcaacgggt gtgcttcccg ttctgatgag tccgtgagga
     1561 cgaaagcgcc tctacaaata attttgttta agagtctatg gactatgttt tcacatatga
     1621 gataccagga 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 cacctgagta gtcaccggct gtgcttgccg gtctgatgag cctgtgaagg
     2581 cgaaactacc tctacaaata attttgttta aacccccgag atgaaataca tcctgtttga
     2641 ggtgtgcgaa atgggtaaaa gccgtgaaca gaccatggaa aatattctga aagcagccaa
     2701 aaagaaattc ggcgaacgtg gttatgaagg caccagcatt caagaaatta ccaaagaagc
     2761 caaagttaac gttgcaatgg ccagctatta ctttaatggc aaagagaacc tgtactacga
     2821 ggtgttcaaa aaatacggtc tggcaaatga actgccgaac tttctggaaa aaaaccagtt
     2881 taatccgatt aatgccctgc gtgaatatct gaccgttttt accacccaca ttaaagaaaa
     2941 tccggaaatt ggcaccctgg cctatgaaga aattatcaaa gaaagcgcac gcctggaaaa
     3001 aatcaaaccg tattttatcg gcagcttcga acagctgaaa gaaattctgc aagagggtga
     3061 aaaacagggt gtgtttcact tttttagcat caaccatacc atccattgga ttaccagcat
     3121 tgttctgttt ccgaaattca aaaaattcat cgatagcctg ggtccgaatg aaaccaatga
     3181 taccaatcat gaatggatgc cggaagatct ggttagccgt attattagcg cactgaccga
     3241 taaaccgaac atttaaaacg catgagaaag cccccggaag atcaccttcc gggggctttt
     3301 ttattgcgca ggtaatggtc accatatatc aagtttacgg ctagctcagt cctaggtact
     3361 atgctagcta ctagagaaag aggagaaata ctagatggct gaagcgcaaa atgatcccct
     3421 gctgccggga tactcgttta atgcccatct ggtggcgggt ttaacgccga ttgaggccaa
     3481 cggttatctc gattttttta tcgaccgacc gctgggaatg aaaggttata ttctcaatct
     3541 caccattcgc ggtcaggggg tggtgaaaaa tcagggacga gaatttgttt gccgaccggg
     3601 tgatattttg ctgttcccgc caggagagat tcatcactac ggtcgtcatc cggaggctcg
     3661 cgaatggtat caccagtggg tttactttcg tccgcgcgcc tactggcatg aatggcttaa
     3721 ctggccgtca atatttgcca atacggggtt ctttcgcccg gatgaagcgc accagccgca
     3781 tttcagcgac ctgtttgggc aaatcattaa cgccgggcaa ggggaagggc gctattcgga
     3841 gctgctggcg ataaatctgc ttgagcaatt gttactgcgg cgcatggaag cgattaacga
     3901 gtcgctccat ccaccgatgg ataatcgggt acgcgaggct tgtcagtaca tcagcgatca
     3961 cctggcagac agcaattttg atatcgccag cgtcgcacag catgtttgct tgtcgccgtc
     4021 gcgtctgtca catcttttcc gccagcagtt agggattagc gtcttaagct ggcgcgagga
     4081 ccaacgtatc agccaggcga agctgctttt gagcaccacc cggatgccta tcgccaccgt
     4141 cggtcgcaat gttggttttg acgatcaact ctatttctcg cgggtattta aaaaatgcac
     4201 cggggccagc ccgagcgagt tccgtgccgg ttaataacca attattgaag gccgctaacg
     4261 cagccttttt ttgtttctgg tctcccaatg gcggcgcgcc atcgaatggc gcaaaacctt
     4321 tcgcggtatg gcatgatagc gcccggaaga gagtcaattc agggtggtga atatgaaacc
     4381 agtaacgtta tacgatgtcg cagagtatgc cggtgtctct tatcagaccg tttcccgcgt
     4441 ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa aaagtggaag cggcgatggc
     4501 ggagctgaat tacattccca accgcgtggc acaacaactg gcgggcaaac agtcgttgct
     4561 gattggcgtt gccacctcca gtctggccct gcacgcgccg tcgcaaattg tcgcggcgat
     4621 taaatctcgc gccgatcaac tgggtgccag cgtggtggtg tcgatggtag aacgaagcgg
     4681 cgtcgaagcc tgtaaagcgg cggtgcacaa tcttctcgcg caacgcgtca gtgggctgat
     4741 cattaactat ccgctggatg accaggatgc cattgctgtg gaagctgcct gcactaatgt
     4801 tccggcgtta tttcttgatg tctctgacca gacacccatc aacagtatta ttttctccca
     4861 tgaggacggt acgcgactgg gcgtggagca tctggtcgca ttgggtcacc agcaaatcgc
     4921 gctgttagcg ggcccattaa gttctgtctc ggcgcgtctg cgtctggctg gctggcataa
     4981 atatctcact cgcaatcaaa ttcagccgat agcggaacgg gaaggcgact ggagtgccat
     5041 gtccggtttt caacaaacca tgcaaatgct gaatgagggc atcgttccca ctgcgatgct
     5101 ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc attaccgagt ccgggctgcg
     5161 cgttggtgcg gatatctcgg tagtgggata cgacgatacc gaagatagct catgttatat
     5221 cccgccgtta accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg
     5281 cttgctgcaa ctctctcagg gccaggcggt gaagggcaat cagctgttgc cagtctcact
     5341 ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     5401 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgacaatc
     5461 caggaggaaa aaaatgtcca gattagataa aagtaaagtg attaacagcg cattagagct
     5521 gcttaatgag gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt
     5581 agagcagcct acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc
     5641 cattgagatg ttagataggc accatactca cttttgccct ttagaagggg aaagctggca
     5701 agatttttta cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg
     5761 agcaaaagta catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca
     5821 attagccttt ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc
     5881 tgtggggcat tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga
     5941 agaaagggaa acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt
     6001 atttgatcac caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg
     6061 attagaaaaa caacttaaat gtgaaagtgg gtcctaaatc acacaggaaa ggcctcgatg
     6121 aaaaacataa atgccgacga cacatacaga ataattaata aaattaaagc ttgtagaagc
     6181 aataatgata ttaatcaatg cttatctgat atgactaaaa tggtacattg tgaatattat
     6241 ttactcgcga tcatttatcc tcattctatg gttaaatctg atatttcaat cctagataat
     6301 taccctaaaa aatggaggca atattatgat gacgctaatt taataaaata tgatcctata
     6361 gtagattatt ctaactccaa tcattcacca attaattgga atatatttga aaacaatgct
     6421 gtaaataaaa aatctccaaa tgtaattaaa gaagcgaaaa catcaggtct tatcactggg
     6481 tttagtttcc ctattcatac ggctaacaat ggcttcggaa tgcttagttt tgcacattca
     6541 gaaaaagaca actatataga tagtttattt ttacatgcgt gtatgaacat accattaatt
     6601 gttccttctc tagttgataa ttatcgaaaa ataaatatag caaataataa atcaaacaac
     6661 gatttaacca aaagagaaaa agaatgttta gcgtgggcat gcgaaggaaa aagctcttgg
     6721 gatatttcaa aaatattagg ttgcagtgag cgtactgtca ctttccattt aaccaatgcg
     6781 caaatgaaac tcaatacaac aaaccgctgc caaagtattt ctaaagcaat tttaacagga
     6841 gcaattgatt gcccatactt taaaaattaa ccataattgg taacgaatca gacaattgac
     6901 ggctcgaggg agtagcatag ggtttgcaga atccctgctt cgtccatttg acaggcacat
     6961 tatgcatcga tgataagctg tcaaacatga gcagatcctc tacgccggac gcatcgtggc
     7021 cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca tcaccgatgg
     7081 ggaagatcgg gctcgccact tcgggctcat gagcaaatat tttatctgag gtgcttcctc
     7141 gctcactgac tcgctgcacg aggcagacct cagcgctagc ggagtgtata ctggcttact
     7201 atgttggcac tgatgagggt gtcagtgaag tgcttcatgt ggcaggagaa aaaaggctgc
     7261 accggtgcgt cagcagaata tgtgatacag gatatattcc gcttcctcgc tcactgactc
     7321 gctacgctcg gtcgttcgac tgcggcgagc ggaaatggct tacgaacggg gcggagattt
     7381 cccggaagat gccaggaaga tacttaacag ggaagtgaga gggccgcggc aaagccgttt
     7441 ttccataggc tccgcccccc tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg
     7501 cgaaacccga caggactata aagataccag gcgtttcccc ctggcggctc cctcgtgcgc
     7561 tctcctgttc ctgcctttcg gtttaccggt gtcattccgc tgttatggcc gcgtttgtct
     7621 cattccacgc ctgacactca gttccgggta ggcagttcgc tccaagctgg actgt
//
