LOCUS       sequence-400057-        7537 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SR latch circuit on pLW555 backbone: PTet-E1, PTac-P1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7537)
  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 7537)
  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: pML3
FEATURES             Location/Qualifiers
     source          1..7537
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     364..383
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     448..467
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     protein_bind    708..726
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    733..751
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        join(1522..1527,1528..1543,1544..1550)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1556..1575
                     /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."
     promoter        2438..2472
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     RBS             2481..2492
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3348..3359
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3477..4559
                     /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(3496..3515)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4578..5201
                     /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(4651..4670)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(6160..6179)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      complement(6544..7089)
                     /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(6624..6643)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7178..7537,1..456))
                     /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(join(7178..7537,1..456))
                     /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"
ORIGIN
        1 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
       61 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
      121 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
      181 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
      241 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
      301 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
      361 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
      421 cctggagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
      481 gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa tgtacatcag
      541 agattttgag acacaaccaa ttattgaagg cctccctaac ggggggcctt tttttgtttc
      601 tggtctcccg cttggagcga cgtacggtgg aatctgattc gttaccaatt gacatgatac
      661 gaaacgtacc gtatcgttaa ggttacgtac tccaccgttg gcttttttcc ctatcagtga
      721 tagagattga catccctatc agtgatagag ataatgagca cctgagaagt caattaatgt
      781 gcttttaatt ctgatgagtc ggtgacgacg aaacttcctc tacaaataat tttgtttaac
      841 cccccgagga gtagcacatg ccgaaactgg gtatgcagag cattcgtcgt cgtcagctga
      901 ttgatgcaac cctggaagca attaatgaag ttggtatgca tgatgcaacc attgcacaga
      961 ttgcacgtcg tgccggtgtt agcaccggta ttattagcca ttatttccgc gataaaaacg
     1021 gtctgctgga agcaaccatg cgtgatatta ccagccagct gcgtgatgca gttctgaatc
     1081 gtctgcatgc actgccgcag ggtagcgcag aacagcgtct gcaggcaatt gttggtggta
     1141 attttgatga aacccaggtt agcagcgcag caatgaaagc atggctggca ttttgggcaa
     1201 gcagcatgca tcagccgatg ctgtatcgtc tgcagcaggt tagcagtcgt cgtctgctga
     1261 gcaatctggt tagcgaattt cgtcgtgaac tgcctcgtga acaggcacaa gaggcaggtt
     1321 atggtctggc agcactgatt gatggtctgt ggctgcgtgc agcactgagc ggtaaaccgc
     1381 tggataaaac ccgtgcaaat agcctgaccc gtcattttat cacccagcat ctgccgaccg
     1441 attaaccaat tattgaacac ccttcggggt gtttttttgt ttctggtcta ccaggtcgct
     1501 ggagaacgat cgttggctgt gttgacaatt aatcatcggc tcgtataatg tgtggaattg
     1561 tgagcgctca caatttacga gcgcgggtga gagggattcg ttaccaatag acaattgatt
     1621 ggacgttcaa tataatgcta gcctgagcgg tcaacgcatg tgctttgcgt tctgatgaga
     1681 cagtgatgtc gaaaccgcct ctacaaataa ttttgtttaa ctatggacta tgtttgaaag
     1741 ggagaaatac tagatggcac gtaccccgag ccgtagcagc attggtagcc tgcgtagtcc
     1801 gcatacccat aaagcaattc tgaccagcac cattgaaatc ctgaaagaat gtggttatag
     1861 cggtctgagc attgaaagcg ttgcacgtcg tgccggtgca agcaaaccga ccatttatcg
     1921 ttggtggacc aataaagcag cactgattgc cgaagtgtat gaaaatgaaa gcgaacaggt
     1981 gcgtaaattt ccggatctgg gtagctttaa agccgatctg gattttctgc tgcgtaatct
     2041 gtggaaagtt tggcgtgaaa ccatttgtgg tgaagcattt cgttgtgtta ttgcagaagc
     2101 acagctggac cctgcaaccc tgacccagct gaaagatcag tttatggaac gtcgtcgtga
     2161 gatgccgaaa aaactggttg aaaatgccat tagcaatggt gaactgccga aagataccaa
     2221 tcgtgaactg ctgctggata tgatttttgg tttttgttgg tatcgcctgc tgaccgaaca
     2281 gctgaccgtt gaacaggata ttgaagaatt taccttcctg ctgattaatg gtgtttgtcc
     2341 gggtacacag cgttaaggaa acacagaaaa aagcccgcac ctgacagtgc gggctttttt
     2401 tttcgaccaa aggaggtaat ggtcaccata tatcaagttt acggctagct cagtcctagg
     2461 tactatgcta gctactagag aaagaggaga aatactagat ggctgaagcg caaaatgatc
     2521 ccctgctgcc gggatactcg tttaatgccc atctggtggc gggtttaacg ccgattgagg
     2581 ccaacggtta tctcgatttt tttatcgacc gaccgctggg aatgaaaggt tatattctca
     2641 atctcaccat tcgcggtcag ggggtggtga aaaatcaggg acgagaattt gtttgccgac
     2701 cgggtgatat tttgctgttc ccgccaggag agattcatca ctacggtcgt catccggagg
     2761 ctcgcgaatg gtatcaccag tgggtttact ttcgtccgcg cgcctactgg catgaatggc
     2821 ttaactggcc gtcaatattt gccaatacgg ggttctttcg cccggatgaa gcgcaccagc
     2881 cgcatttcag cgacctgttt gggcaaatca ttaacgccgg gcaaggggaa gggcgctatt
     2941 cggagctgct ggcgataaat ctgcttgagc aattgttact gcggcgcatg gaagcgatta
     3001 acgagtcgct ccatccaccg atggataatc gggtacgcga ggcttgtcag tacatcagcg
     3061 atcacctggc agacagcaat tttgatatcg ccagcgtcgc acagcatgtt tgcttgtcgc
     3121 cgtcgcgtct gtcacatctt ttccgccagc agttagggat tagcgtctta agctggcgcg
     3181 aggaccaacg tatcagccag gcgaagctgc ttttgagcac cacccggatg cctatcgcca
     3241 ccgtcggtcg caatgttggt tttgacgatc aactctattt ctcgcgggta tttaaaaaat
     3301 gcaccggggc cagcccgagc gagttccgtg ccggttaata accaattatt gaaggccgct
     3361 aacgcagcct ttttttgttt ctggtctccc aatggcggcg cgccatcgaa tggcgcaaaa
     3421 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatatga
     3481 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     3541 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     3601 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     3661 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     3721 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     3781 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     3841 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     3901 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     3961 cccatgagga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     4021 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     4081 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     4141 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     4201 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     4261 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagat agctcatgtt
     4321 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     4381 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgccagtct
     4441 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     4501 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgac
     4561 aatccaggag gaaaaaaatg tccagattag ataaaagtaa agtgattaac agcgcattag
     4621 agctgcttaa tgaggtcgga atcgaaggtt taacaacccg taaactcgcc cagaagctag
     4681 gtgtagagca gcctacattg tattggcatg taaaaaataa gcgggctttg ctcgacgcct
     4741 tagccattga gatgttagat aggcaccata ctcacttttg ccctttagaa ggggaaagct
     4801 ggcaagattt tttacgtaat aacgctaaaa gttttagatg tgctttacta agtcatcgcg
     4861 atggagcaaa agtacattta ggtacacggc ctacagaaaa acagtatgaa actctcgaaa
     4921 atcaattagc ctttttatgc caacaaggtt tttcactaga gaatgcatta tatgcactca
     4981 gcgctgtggg gcattttact ttaggttgcg tattggaaga tcaagagcat caagtcgcta
     5041 aagaagaaag ggaaacacct actactgata gtatgccgcc attattacga caagctatcg
     5101 aattatttga tcaccaaggt gcagagccag ccttcttatt cggccttgaa ttgatcatat
     5161 gcggattaga aaaacaactt aaatgtgaaa gtgggtccta aatcacacag gaaaggcctc
     5221 gatgaaaaac ataaatgccg acgacacata cagaataatt aataaaatta aagcttgtag
     5281 aagcaataat gatattaatc aatgcttatc tgatatgact aaaatggtac attgtgaata
     5341 ttatttactc gcgatcattt atcctcattc tatggttaaa tctgatattt caatcctaga
     5401 taattaccct aaaaaatgga ggcaatatta tgatgacgct aatttaataa aatatgatcc
     5461 tatagtagat tattctaact ccaatcattc accaattaat tggaatatat ttgaaaacaa
     5521 tgctgtaaat aaaaaatctc caaatgtaat taaagaagcg aaaacatcag gtcttatcac
     5581 tgggtttagt ttccctattc atacggctaa caatggcttc ggaatgctta gttttgcaca
     5641 ttcagaaaaa gacaactata tagatagttt atttttacat gcgtgtatga acataccatt
     5701 aattgttcct tctctagttg ataattatcg aaaaataaat atagcaaata ataaatcaaa
     5761 caacgattta accaaaagag aaaaagaatg tttagcgtgg gcatgcgaag gaaaaagctc
     5821 ttgggatatt tcaaaaatat taggttgcag tgagcgtact gtcactttcc atttaaccaa
     5881 tgcgcaaatg aaactcaata caacaaaccg ctgccaaagt atttctaaag caattttaac
     5941 aggagcaatt gattgcccat actttaaaaa ttaaccataa ttggtaacga atcagacaat
     6001 tgacggctcg agggagtagc atagggtttg cagaatccct gcttcgtcca tttgacaggc
     6061 acattatgca tcgatgataa gctgtcaaac atgagcagat cctctacgcc ggacgcatcg
     6121 tggccggcat caccggcgcc acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg
     6181 atggggaaga tcgggctcgc cacttcgggc tcatgagcaa atattttatc tgaggtgctt
     6241 cctcgctcac tgactcgctg cacgaggcag acctcagcgc tagcggagtg tatactggct
     6301 tactatgttg gcactgatga gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg
     6361 ctgcaccggt gcgtcagcag aatatgtgat acaggatata ttccgcttcc tcgctcactg
     6421 actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag
     6481 atttcccgga agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc
     6541 gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg
     6601 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt
     6661 gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt
     6721 gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta
     6781 tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6841 ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag
     6901 gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac
     6961 tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa
     7021 aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa
     7081 cgatctcaag aagatcatct tattaagggg tctgacgctc agtggaacga aaaatcaatc
     7141 taaagtatat atgagtaaac ttggtctgac agttacctta gaaaaactca tcgagcatca
     7201 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     7261 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     7321 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     7381 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     7441 gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     7501 cactcgcatc aaccaaaccg ttattcattc gtgattg
//
