LOCUS       sequence-400062-        7567 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SR latch circuit on pLW555 backbone: PTac-P3, PTet-H1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7567)
  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 7567)
  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: pML5
FEATURES             Location/Qualifiers
     source          1..7567
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     39..58
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     123..142
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        join(310..315,316..331,332..338)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    344..363
                     /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    1289..1307
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1314..1332
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        2143..2177
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     RBS             2186..2197
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             3053..3064
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             3182..4264
                     /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(3201..3220)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4283..4906
                     /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(4356..4375)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5865..5884)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      complement(6249..6794)
                     /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(6329..6348)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6883..7567,1..131))
                     /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(6883..7567,1..131))
                     /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 tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca tttataccca
       61 tataaatcag catccatgtt ggaatttaat cgcggcctgg agcaagacgt ttcccgttga
      121 atatggctca taacacccct tgtattactg tttatgtaag cagacagttt tattgttcat
      181 gatgatatat ttttatcttg tgcaatgtac atcagagatt ttgagacaca accaattatt
      241 gaaggcctcc ctaacggggg gccttttttt gtttctggtc tcccgcttgg agaacgatcg
      301 ttggctgtgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg agcgctcaca
      361 atttacgagc aggaatctga acgattcgtt accaattgcc atatttaaaa ttcttgttta
      421 aaatgctagc ctgagcggtc aacgcatgtg ctttgcgttc tgatgagaca gtgatgtcga
      481 aaccgcctct acaaataatt ttgtttaact ttacgagggc gatcctatgg cacgtacccc
      541 gagccgtagc agcattggta gcctgcgtag tccgcatacc cataaagcaa ttctgaccag
      601 caccattgaa atcctgaaag aatgtggtta tagcggtctg agcattgaaa gcgttgcacg
      661 tcgtgccggt gcaagcaaac cgaccattta tcgttggtgg accaataaag cagcactgat
      721 tgccgaagtg tatgaaaatg aaagcgaaca ggtgcgtaaa tttccggatc tgggtagctt
      781 taaagccgat ctggattttc tgctgcgtaa tctgtggaaa gtttggcgtg aaaccatttg
      841 tggtgaagca tttcgttgtg ttattgcaga agcacagctg gaccctgcaa ccctgaccca
      901 gctgaaagat cagtttatgg aacgtcgtcg tgagatgccg aaaaaactgg ttgaaaatgc
      961 cattagcaat ggtgaactgc cgaaagatac caatcgtgaa ctgctgctgg atatgatttt
     1021 tggtttttgt tggtatcgcc tgctgaccga acagctgacc gttgaacagg atattgaaga
     1081 atttaccttc ctgctgatta atggtgtttg tccgggtaca cagcgttaag gaaacacaga
     1141 aaaaagcccg cacctgacag tgcgggcttt ttttttcgac caaaggaggt cgctggagcg
     1201 acgtacggtg gaatctgatt cgttaccaat tgacatgata cgaaacgtac cgtatcgtta
     1261 aggttacgta ctccaccgtt ggcttttttc cctatcagtg atagagattg acatccctat
     1321 cagtgataga gataatgagc acctgagtag tcaccggctg tgcttgccgg tctgatgagc
     1381 ctgtgaaggc gaaactacct ctacaaataa ttttgtttaa acccccgaga tgaaatacat
     1441 cctgtttgag gtgtgcgaaa tgggtaaaag ccgtgaacag accatggaaa atattctgaa
     1501 agcagccaaa aagaaattcg gcgaacgtgg ttatgaaggc accagcattc aagaaattac
     1561 caaagaagcc aaagttaacg ttgcaatggc cagctattac tttaatggca aagagaacct
     1621 gtactacgag gtgttcaaaa aatacggtct ggcaaatgaa ctgccgaact ttctggaaaa
     1681 aaaccagttt aatccgatta atgccctgcg tgaatatctg accgttttta ccacccacat
     1741 taaagaaaat ccggaaattg gcaccctggc ctatgaagaa attatcaaag aaagcgcacg
     1801 cctggaaaaa atcaaaccgt attttatcgg cagcttcgaa cagctgaaag aaattctgca
     1861 agagggtgaa aaacagggtg tgtttcactt ttttagcatc aaccatacca tccattggat
     1921 taccagcatt gttctgtttc cgaaattcaa aaaattcatc gatagcctgg gtccgaatga
     1981 aaccaatgat accaatcatg aatggatgcc ggaagatctg gttagccgta ttattagcgc
     2041 actgaccgat aaaccgaaca tttaaaacgc atgagaaagc ccccggaaga tcaccttccg
     2101 ggggcttttt tattgcgcag gtaatggtca ccatatatca agtttacggc tagctcagtc
     2161 ctaggtacta tgctagctac tagagaaaga ggagaaatac tagatggctg aagcgcaaaa
     2221 tgatcccctg ctgccgggat actcgtttaa tgcccatctg gtggcgggtt taacgccgat
     2281 tgaggccaac ggttatctcg atttttttat cgaccgaccg ctgggaatga aaggttatat
     2341 tctcaatctc accattcgcg gtcagggggt ggtgaaaaat cagggacgag aatttgtttg
     2401 ccgaccgggt gatattttgc tgttcccgcc aggagagatt catcactacg gtcgtcatcc
     2461 ggaggctcgc gaatggtatc accagtgggt ttactttcgt ccgcgcgcct actggcatga
     2521 atggcttaac tggccgtcaa tatttgccaa tacggggttc tttcgcccgg atgaagcgca
     2581 ccagccgcat ttcagcgacc tgtttgggca aatcattaac gccgggcaag gggaagggcg
     2641 ctattcggag ctgctggcga taaatctgct tgagcaattg ttactgcggc gcatggaagc
     2701 gattaacgag tcgctccatc caccgatgga taatcgggta cgcgaggctt gtcagtacat
     2761 cagcgatcac ctggcagaca gcaattttga tatcgccagc gtcgcacagc atgtttgctt
     2821 gtcgccgtcg cgtctgtcac atcttttccg ccagcagtta gggattagcg tcttaagctg
     2881 gcgcgaggac caacgtatca gccaggcgaa gctgcttttg agcaccaccc ggatgcctat
     2941 cgccaccgtc ggtcgcaatg ttggttttga cgatcaactc tatttctcgc gggtatttaa
     3001 aaaatgcacc ggggccagcc cgagcgagtt ccgtgccggt taataaccaa ttattgaagg
     3061 ccgctaacgc agcctttttt tgtttctggt ctcccaatgg cggcgcgcca tcgaatggcg
     3121 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     3181 tatgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     3241 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     3301 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     3361 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     3421 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     3481 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     3541 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     3601 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     3661 tttctcccat gaggacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     3721 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     3781 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     3841 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     3901 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     3961 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagatagctc
     4021 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     4081 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     4141 agtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     4201 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     4261 gtgacaatcc aggaggaaaa aaatgtccag attagataaa agtaaagtga ttaacagcgc
     4321 attagagctg cttaatgagg tcggaatcga aggtttaaca acccgtaaac tcgcccagaa
     4381 gctaggtgta gagcagccta cattgtattg gcatgtaaaa aataagcggg ctttgctcga
     4441 cgccttagcc attgagatgt tagataggca ccatactcac ttttgccctt tagaagggga
     4501 aagctggcaa gattttttac gtaataacgc taaaagtttt agatgtgctt tactaagtca
     4561 tcgcgatgga gcaaaagtac atttaggtac acggcctaca gaaaaacagt atgaaactct
     4621 cgaaaatcaa ttagcctttt tatgccaaca aggtttttca ctagagaatg cattatatgc
     4681 actcagcgct gtggggcatt ttactttagg ttgcgtattg gaagatcaag agcatcaagt
     4741 cgctaaagaa gaaagggaaa cacctactac tgatagtatg ccgccattat tacgacaagc
     4801 tatcgaatta tttgatcacc aaggtgcaga gccagccttc ttattcggcc ttgaattgat
     4861 catatgcgga ttagaaaaac aacttaaatg tgaaagtggg tcctaaatca cacaggaaag
     4921 gcctcgatga aaaacataaa tgccgacgac acatacagaa taattaataa aattaaagct
     4981 tgtagaagca ataatgatat taatcaatgc ttatctgata tgactaaaat ggtacattgt
     5041 gaatattatt tactcgcgat catttatcct cattctatgg ttaaatctga tatttcaatc
     5101 ctagataatt accctaaaaa atggaggcaa tattatgatg acgctaattt aataaaatat
     5161 gatcctatag tagattattc taactccaat cattcaccaa ttaattggaa tatatttgaa
     5221 aacaatgctg taaataaaaa atctccaaat gtaattaaag aagcgaaaac atcaggtctt
     5281 atcactgggt ttagtttccc tattcatacg gctaacaatg gcttcggaat gcttagtttt
     5341 gcacattcag aaaaagacaa ctatatagat agtttatttt tacatgcgtg tatgaacata
     5401 ccattaattg ttccttctct agttgataat tatcgaaaaa taaatatagc aaataataaa
     5461 tcaaacaacg atttaaccaa aagagaaaaa gaatgtttag cgtgggcatg cgaaggaaaa
     5521 agctcttggg atatttcaaa aatattaggt tgcagtgagc gtactgtcac tttccattta
     5581 accaatgcgc aaatgaaact caatacaaca aaccgctgcc aaagtatttc taaagcaatt
     5641 ttaacaggag caattgattg cccatacttt aaaaattaac cataattggt aacgaatcag
     5701 acaattgacg gctcgaggga gtagcatagg gtttgcagaa tccctgcttc gtccatttga
     5761 caggcacatt atgcatcgat gataagctgt caaacatgag cagatcctct acgccggacg
     5821 catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat
     5881 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcaaatatt ttatctgagg
     5941 tgcttcctcg ctcactgact cgctgcacga ggcagacctc agcgctagcg gagtgtatac
     6001 tggcttacta tgttggcact gatgagggtg tcagtgaagt gcttcatgtg gcaggagaaa
     6061 aaaggctgca ccggtgcgtc agcagaatat gtgatacagg atatattccg cttcctcgct
     6121 cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg
     6181 cggagatttc ccggaagatg ccaggaagat acttaacagg gaagtgagag ggccgcggca
     6241 aagccgtttt tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat
     6301 cagtggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggcggctcc
     6361 ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg tcattccgct gttatggccg
     6421 cgtttgtctc attccacgcc tgacactcag ttccgggtag gcagttcgct ccaagctgga
     6481 ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc ttatccggta actatcgtct
     6541 tgagtccaac ccggaaagac atgcaaaagc accactggca gcagccactg gtaattgatt
     6601 tagaggagtt agtcttgaag tcatgcgccg gttaaggcta aactgaaagg acaagttttg
     6661 gtgactgcgc tcctccaagc cagttacctc ggttcaaaga gttggtagct cagagaacct
     6721 tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga gcaagagatt acgcgcagac
     6781 caaaacgatc tcaagaagat catcttatta aggggtctga cgctcagtgg aacgaaaaat
     6841 caatctaaag tatatatgag taaacttggt ctgacagtta ccttagaaaa actcatcgag
     6901 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     6961 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     7021 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     7081 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     7141 caaaagctta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     7201 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     7261 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     7321 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     7381 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     7441 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     7501 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     7561 cttccca
//
