LOCUS       sequence-400060-        5305 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid backbone for choline sensing circuits. The plasmid contains,
            lacI and tetR as well as betIM. The plasmid also contains the p15a 
            ori.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5305)
  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 5305)
  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: pML523
FEATURES             Location/Qualifiers
     source          1..5305
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     866..885
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(1068..1691)
                     /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     1599..1618
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     CDS             complement(1710..2792)
                     /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     2754..2773
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    2971..2992
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3007..3012,3013..3030,3031..3037)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3045..3061
                     /label=lac operator
                     /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)."
     primer_bind     3050..3072
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3069..3085
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3069..3085
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_feature    3098..3154
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(3155..3172)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3155..3171)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3164..3186)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3527..3546)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3538..4353
                     /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             3538..4353
                     /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     complement(3611..3630)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4442..4987
                     /direction=RIGHT
                     /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     4888..4907
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5104..5121
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 aaaaaaacac cccgaagggt gttcaataat tggttaatcg gtcggcagat gctgggtgat
       61 aaaatgacgg gtcaggctat ttgcacgggt tttatccagc ggtttaccgc tcagtgctgc
      121 acgcagccac agaccatcaa tcagtgctgc cagaccataa cctgcctctt gtgcctgttc
      181 acgaggcagt tcacgacgaa attcgctaac cagattgctc agcagacgac gactgctaac
      241 ctgctgcaga cgatacagca tcggctgatg catgctgatt gcccaaaatg ccagccatgc
      301 tttcattgct gcgctgctaa cctgggtttc atcaaaatta ccaccaacaa ttgcctgcag
      361 acgctgttct gcgctaccct gcggcagtgc atgcagacga ttcagaactg catcacgcag
      421 ctggctggta atatcacgca tggttgcttc cagcagaccg tttttatcgc ggaaataatg
      481 gctaataata ccggtgctaa caccggcacg acgtgcaatc tgtgcaatgg ttgcatcatg
      541 cataccaact tcattaattg cttccagggt tgcatcaatc agctgacgac gacgaatgct
      601 ctgcataccc agtttcggca tgtgctactc ctcggggggt taaacaaaat tatttgtaga
      661 ggaagtttcg tcgtcaccga ctcatcagaa ttaaaagcac attaattgac ttctcagtgc
      721 tagcattata ccactatatc ctagttgtgt caaagttagg gacttatcct ccagtatcgt
      781 cctctctgct ctgtgtgagc gaggaagcac ctcagataaa atatttgctc atgagcccga
      841 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
      901 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatctgc tcatgtttga
      961 cagcttatca tcgatgcata atgtgcctgt caaatggacg aagcagggat tctgcaaacc
     1021 ctatgctact ccctcgagcc gtcaattgtc tgattcgtta ccaattatta ggacccactt
     1081 tcacatttaa gttgtttttc taatccgcat atgatcaatt caaggccgaa taagaaggct
     1141 ggctctgcac cttggtgatc aaataattcg atagcttgtc gtaataatgg cggcatacta
     1201 tcagtagtag gtgtttccct ttcttcttta gcgacttgat gctcttgatc ttccaatacg
     1261 caacctaaag taaaatgccc cacagcgctg agtgcatata atgcattctc tagtgaaaaa
     1321 ccttgttggc ataaaaaggc taattgattt tcgagagttt catactgttt ttctgtaggc
     1381 cgtgtaccta aatgtacttt tgctccatcg cgatgactta gtaaagcaca tctaaaactt
     1441 ttagcgttat tacgtaaaaa atcttgccag ctttcccctt ctaaagggca aaagtgagta
     1501 tggtgcctat ctaacatctc aatggctaag gcgtcgagca aagcccgctt attttttaca
     1561 tgccaataca atgtaggctg ctctacacct agcttctggg cgagtttacg ggttgttaaa
     1621 ccttcgattc cgacctcatt aagcagctct aatgcgctgt taatcacttt acttttatct
     1681 aatctggaca tttttttcct cctggattgt cactgcccgc tttccagtcg ggaaacctgt
     1741 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     1801 gccagggtgg tttttctttt caccagtgag actggcaaca gctgattgcc cttcaccgcc
     1861 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     1921 tgtttgatgg tggttaacgg cgggatataa catgagctat cttcggtatc gtcgtatccc
     1981 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     2041 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     2101 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     2161 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     2221 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     2281 acgcccagtc gcgtaccgtc ctcatgggag aaaataatac tgttgatggg tgtctggtca
     2341 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     2401 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     2461 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     2521 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     2581 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     2641 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     2701 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     2761 tctgcgacat cgtataacgt tactggtttc atattcacca ccctgaattg actctcttcc
     2821 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggcgcg ccgccattca
     2881 ttctgtcttc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
     2941 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
     3001 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
     3061 atttcacaca ggaaacagct atgaccatga ttacgccaag cttgcatgcc tgcaggtcga
     3121 ctctagagga tccccgggta ccgagctcga attcactggc cgtcgtttta caacgtcgtg
     3181 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     3241 gctggcgtaa tagcgaagag gcccacaccg atcgcccttc ccaacagttg cgcagcctga
     3301 atggctaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3361 gcatatggtg cagaagacag aagcgggaga ccagaaacaa aaaaaggccc cccgttaggg
     3421 aggccttcaa taattggttg tgtctcaaaa tctctgatgt acattgcaca agataaaaat
     3481 atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
     3541 agccatattc aacgggaaac gtcttgctcc aggccgcgat taaattccaa catggatgct
     3601 gatttatatg ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat
     3661 cgattgtatg ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt
     3721 gccaatgatg ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt
     3781 ccgaccatca agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc
     3841 cccgggaaaa cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt
     3901 gatgcgctgg cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt
     3961 aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt
     4021 gatgcgagtg attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa
     4081 atgcataagc ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt
     4141 gataacctta tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga
     4201 atcgcagacc gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct
     4261 tcattacaga aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg
     4321 cagtttcatt tgatgctcga tgagtttttc taaggtaact gtcagaccaa gtttactcat
     4381 atatacttta gattgatttt tcgttccact gagcgtcaga ccccttaata agatgatctt
     4441 cttgagatcg ttttggtctg cgcgtaatct cttgctctga aaacgaaaaa accgccttgc
     4501 agggcggttt ttcgaaggtt ctctgagcta ccaactcttt gaaccgaggt aactggcttg
     4561 gaggagcgca gtcaccaaaa cttgtccttt cagtttagcc ttaaccggcg catgacttca
     4621 agactaactc ctctaaatca attaccagtg gctgctgcca gtggtgcttt tgcatgtctt
     4681 tccgggttgg actcaagacg atagttaccg gataaggcgc agcggtcgga ctgaacgggg
     4741 ggttcgtgca tacagtccag cttggagcga actgcctacc cggaactgag tgtcaggcgt
     4801 ggaatgagac aaacgcggcc ataacagcgg aatgacaccg gtaaaccgaa aggcaggaac
     4861 aggagagcgc acgagggagc cgccaggggg aaacgcctgg tatctttata gtcctgtcgg
     4921 gtttcgccac cactgatttg agcgtcagat ttcgtgatgc ttgtcagggg ggcggagcct
     4981 atggaaaaac ggctttgccg cggccctctc acttccctgt taagtatctt cctggcatct
     5041 tccgggaaat ctccgccccg ttcgtaagcc atttccgctc gccgcagtcg aacgaccgag
     5101 cgtagcgagt cagtgagcga ggaagcggaa tatatcctgt atcacatatt ctgctgacgc
     5161 accggtgcag ccttttttct cctgccacat gaagcacttc actgacaccc tcatcagtgc
     5221 caacatagta agccagtata cactccgcta gcgctgaggt ctgccgattc tgcctggaga
     5281 cctggcaacc tggtagacca gaaac
//
