LOCUS       sequence-400063-        6187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid backbone for gamma-aminobutyric acid circuits. The plasmid 
            contains gabR, lacI and tetR as well as the resistance gene kanR and
            the p15a ori..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6187)
  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 6187)
  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: pML3001
FEATURES             Location/Qualifiers
     source          1..6187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             123..134
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(342..359)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(476..1021)
                     /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(556..575)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1110..1925)
                     /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(1110..1925)
                     /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     1833..1852
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1917..1936
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     2277..2299
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2291..2308
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2292..2308
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    2309..2365
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(2378..2394)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2378..2394)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2391..2413)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2402..2418
                     /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)."
     promoter        complement(join(2426..2432,2433..2450,2451..2456))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2471..2492
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             2671..3753
                     /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(2690..2709)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3772..4395
                     /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(3845..3864)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(4578..4597)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4695..4729
                     /label=BBa_J23106
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.47"
ORIGIN
        1 tgctctcatt atcggctttg cacggctgaa ggaagaagat attcaggagg gtgtgcagcg
       61 gcttttcaaa gcggtttacg gacataaaaa aatccccgtt acaggggatt gactgaccaa
      121 ttattgaagg ccgctaacgc ggcctttttt tgtttctggt cacccatcgg caggttgcca
      181 ggtctccagg cagaatcggc agacctcagc gctagcggag tgtatactgg cttactatgt
      241 tggcactgat gagggtgtca gtgaagtgct tcatgtggca ggagaaaaaa ggctgcaccg
      301 gtgcgtcagc agaatatgtg atacaggata tattccgctt cctcgctcac tgactcgcta
      361 cgctcggtcg ttcgactgcg gcgagcggaa atggcttacg aacggggcgg agatttcccg
      421 gaagatgcca ggaagatact taacagggaa gtgagagggc cgcggcaaag ccgtttttcc
      481 ataggctccg cccccctgac aagcatcacg aaatctgacg ctcaaatcag tggtggcgaa
      541 acccgacagg actataaaga taccaggcgt ttccccctgg cggctccctc gtgcgctctc
      601 ctgttcctgc ctttcggttt accggtgtca ttccgctgtt atggccgcgt ttgtctcatt
      661 ccacgcctga cactcagttc cgggtaggca gttcgctcca agctggactg tatgcacgaa
      721 ccccccgttc agtccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
      781 gaaagacatg caaaagcacc actggcagca gccactggta attgatttag aggagttagt
      841 cttgaagtca tgcgccggtt aaggctaaac tgaaaggaca agttttggtg actgcgctcc
      901 tccaagccag ttacctcggt tcaaagagtt ggtagctcag agaaccttcg aaaaaccgcc
      961 ctgcaaggcg gttttttcgt tttcagagca agagattacg cgcagaccaa aacgatctca
     1021 agaagatcat cttattaagg ggtctgacgc tcagtggaac gaaaaatcaa tctaaagtat
     1081 atatgagtaa acttggtctg acagttacct tagaaaaact catcgagcat caaatgaaac
     1141 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     1201 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     1261 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     1321 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagcttatgc
     1381 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     1441 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     1501 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     1561 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     1621 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     1681 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     1741 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     1801 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     1861 tcagcatcca tgttggaatt taatcgcggc ctggagcaag acgtttcccg ttgaatatgg
     1921 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgatgat
     1981 atatttttat cttgtgcaat gtacatcaga gattttgaga cacaaccaat tattgaaggc
     2041 ctccctaacg gggggccttt ttttgtttct ggtctcccgc ttctgtcttc tgcaccatat
     2101 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattagcc
     2161 attcaggctg cgcaactgtt gggaagggcg atcggtgtgg gcctcttcgc tattacgcca
     2221 gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca
     2281 gtcacgacgt tgtaaaacga cggccagtga attcgagctc ggtacccggg gatcctctag
     2341 agtcgacctg caggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     2401 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     2461 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     2521 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggaagacag
     2581 aatgaatggc ggcgcgccat cgaatggcgc aaaacctttc gcggtatggc atgatagcgc
     2641 ccggaagaga gtcaattcag ggtggtgaat atgaaaccag taacgttata cgatgtcgca
     2701 gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt
     2761 tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac
     2821 cgcgtggcac aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt
     2881 ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg
     2941 ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg
     3001 gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac
     3061 caggatgcca ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc
     3121 tctgaccaga cacccatcaa cagtattatt ttctcccatg aggacggtac gcgactgggc
     3181 gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt
     3241 tctgtctcgg cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt
     3301 cagccgatag cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg
     3361 caaatgctga atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg
     3421 ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatctcggta
     3481 gtgggatacg acgataccga agatagctca tgttatatcc cgccgttaac caccatcaaa
     3541 caggattttc gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc
     3601 caggcggtga agggcaatca gctgttgcca gtctcactgg tgaaaagaaa aaccaccctg
     3661 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     3721 cgacaggttt cccgactgga aagcgggcag tgacaatcca ggaggaaaaa aatgtccaga
     3781 ttagataaaa gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa
     3841 ggtttaacaa cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg
     3901 catgtaaaaa ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac
     3961 catactcact tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct
     4021 aaaagtttta gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca
     4081 cggcctacag aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa
     4141 ggtttttcac tagagaatgc attatatgca ctcagcgctg tggggcattt tactttaggt
     4201 tgcgtattgg aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact
     4261 gatagtatgc cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag
     4321 ccagccttct tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt
     4381 gaaagtgggt cctaataatt ggtaacgaat cagacaattg acggctcgag ggagtagcat
     4441 agggtttgca gaatccctgc ttcgtccatt tgacaggcac attatgcatc gatgataagc
     4501 tgtcaaacat gagcagatcc tctacgccgg acgcatcgtg gccggcatca ccggcgccac
     4561 aggtgcggtt gctggcgcct atatcgccga catcaccgat ggggaagatc gggctcgcca
     4621 cttcgggctc atgagcaaat attttatctg aggtgcttcc tcgctcacac agagcagaga
     4681 ggacgatact ggagtttacg gctagctcag tcctaggtat agtgctagcc tgagtccgag
     4741 tgcttacagt tctgaaggag tcaattaatg tgcttttaat tctgatgaga cggtgacgtc
     4801 gaaactccct ctacaaataa ttttgtttaa taacatgttg tcaatgagga gctatcttga
     4861 tggatatcac gattacactc gatcgttcag aacaagccga ttatatctat cagcaaattt
     4921 atcaaaagct gaaaaaagaa atcctcagcc gcaatctgct gccgcactcg aaggttccct
     4981 ccaagcggga gctggctgaa aatctcaagg tcagcgtaaa ttcagtgaat tcagcctatc
     5041 agcagctgct ggctgagggg tatttgtacg ccattgaacg aaagggtttc ttcgtggagg
     5101 aactagacat gttttccgcc gaggagcacc ctccatttgc actgccggat gacctaaaag
     5161 agattcacat cgaccagagc gattggatat cgttttcaca catgagttcc gatacagacc
     5221 attttccgat caaaagctgg ttccgctgcg agcaaaaagc ggcctcccgc tcataccgca
     5281 cgctcggcga tatgtcacat ccgcaaggga tatatgaagt gagagcggcc attacgaggc
     5341 tcatttccct gacgaggggt gtaaaatgca ggccggaaca aatgatcata ggggcaggca
     5401 cacaggtgct catgcagctg ttgactgagc ttttacccaa ggaagccgtg tatgcgatgg
     5461 aggagcctgg ctacaggcgc atgtatcagc ttttgaagaa tgccggaaaa caagtaaaga
     5521 cgatcatgct ggatgaaaaa ggcatgtcga ttgctgaaat caccagacag cagccagatg
     5581 tgctggtgac caccccgtcg catcagtttc cgtccggaac gattatgcct gtatccagaa
     5641 gaattcagct gctgaactgg gcagccgagg agccgcgccg atatatcatt gaggacgatt
     5701 atgatagtga attcacatat gatgtagaca gtattccggc gctgcaaagc ctcgaccgtt
     5761 ttcaaaatgt catctatatg ggaacctttt caaagtccct tctccccggc ttacggatca
     5821 gctatatggt gttgccgcct gagctgttga gggcatacaa acagcggggc tatgatctgc
     5881 agacttgctc atcactcaca cagctcaccc tgcaggaatt tatcgagtct ggtgaatatc
     5941 agaagcatat aaaaaaaatg aagcagcatt ataaagaaaa gagagaacgc ctgatcaccg
     6001 ctttagaagc agagttcagc ggagaggtta ccgtaaaagg ggcaaatgcg gggctgcatt
     6061 ttgttaccga atttgatacc aggcgcaccg aacaagacat cctgtcacat gctgccgggc
     6121 tgcagcttga aatattcgga atgagccgat ttaacttgaa ggaaaacaag cggcaaacgg
     6181 gcaggcc
//
