LOCUS       sequence-362681-        5455 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Circuit backbone plasmid (LacZa insert).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5455)
  AUTHORS   Nielsen AA, Der BS, Shin J, Vaidyanathan P, Paralanov V, Strychalski
            EA, Ross D, Densmore D, Voigt CA
  TITLE     Genetic circuit design automation.
  JOURNAL   Science. 2016 Apr 1;352(6281):aac7341. doi: 10.1126/science.aac7341.
  PUBMED    27034378
REFERENCE   2  (bases 1 to 5455)
  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: "Science."; 
            date: "2016-04-1"; volume: "352(6281):aac7341. doi"; pages: " 
            10.1126/science.aac7341"
COMMENT     Sequence Label: pAN1201
FEATURES             Location/Qualifiers
     source          1..5455
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1..57
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(70..86)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(70..86)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(83..105)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    94..110
                     /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(118..124,125..142,143..148))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    163..184
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        293..327
                     /label=BBa_J23105
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 0.24"
     RBS             336..347
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             1203..1214
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1332..2414
                     /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(1351..1370)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             2433..3056
                     /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(2506..2525)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(3239..3258)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     rep_origin      complement(3623..4168)
                     /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(3703..3722)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4257..5072)
                     /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(4257..5072)
                     /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     4980..4999
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5064..5083
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     5424..5446
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5439..5455
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gaattcgagc tcggtacccg gggatcctct agagtcgacc tgcaggcatg caagcttggc
       61 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
      121 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
      181 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
      241 ttaatgaatc ggccaacgcg cggggaagac agaatggtca ccatatatca agtttacggc
      301 tagctcagtc ctaggtacta tgctagctac tagagaaaga ggagaaatac tagatggctg
      361 aagcgcaaaa tgatcccctg ctgccgggat actcgtttaa tgcccatctg gtggcgggtt
      421 taacgccgat tgaggccaac ggttatctcg atttttttat cgaccgaccg ctgggaatga
      481 aaggttatat tctcaatctc accattcgcg gtcagggggt ggtgaaaaat cagggacgag
      541 aatttgtttg ccgaccgggt gatattttgc tgttcccgcc aggagagatt catcactacg
      601 gtcgtcatcc ggaggctcgc gaatggtatc accagtgggt ttactttcgt ccgcgcgcct
      661 actggcatga atggcttaac tggccgtcaa tatttgccaa tacggggttc tttcgcccgg
      721 atgaagcgca ccagccgcat ttcagcgacc tgtttgggca aatcattaac gccgggcaag
      781 gggaagggcg ctattcggag ctgctggcga taaatctgct tgagcaattg ttactgcggc
      841 gcatggaagc gattaacgag tcgctccatc caccgatgga taatcgggta cgcgaggctt
      901 gtcagtacat cagcgatcac ctggcagaca gcaattttga tatcgccagc gtcgcacagc
      961 atgtttgctt gtcgccgtcg cgtctgtcac atcttttccg ccagcagtta gggattagcg
     1021 tcttaagctg gcgcgaggac caacgtatca gccaggcgaa gctgcttttg agcaccaccc
     1081 ggatgcctat cgccaccgtc ggtcgcaatg ttggttttga cgatcaactc tatttctcgc
     1141 gggtatttaa aaaatgcacc ggggccagcc cgagcgagtt ccgtgccggt taataaccaa
     1201 ttattgaagg ccgctaacgc agcctttttt tgtttctggt ctcccaatgg cggcgcgcca
     1261 tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
     1321 gggtggtgaa tatgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
     1381 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
     1441 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
     1501 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
     1561 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
     1621 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
     1681 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
     1741 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
     1801 acagtattat tttctcccat gaggacggta cgcgactggg cgtggagcat ctggtcgcat
     1861 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
     1921 gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg
     1981 aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca
     2041 tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca
     2101 ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg
     2161 aagatagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg
     2221 ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc
     2281 agctgttgcc agtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg
     2341 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     2401 aaagcgggca gtgacaatcc aggaggaaaa aaatgtccag attagataaa agtaaagtga
     2461 ttaacagcgc attagagctg cttaatgagg tcggaatcga aggtttaaca acccgtaaac
     2521 tcgcccagaa gctaggtgta gagcagccta cattgtattg gcatgtaaaa aataagcggg
     2581 ctttgctcga cgccttagcc attgagatgt tagataggca ccatactcac ttttgccctt
     2641 tagaagggga aagctggcaa gattttttac gtaataacgc taaaagtttt agatgtgctt
     2701 tactaagtca tcgcgatgga gcaaaagtac atttaggtac acggcctaca gaaaaacagt
     2761 atgaaactct cgaaaatcaa ttagcctttt tatgccaaca aggtttttca ctagagaatg
     2821 cattatatgc actcagcgct gtggggcatt ttactttagg ttgcgtattg gaagatcaag
     2881 agcatcaagt cgctaaagaa gaaagggaaa cacctactac tgatagtatg ccgccattat
     2941 tacgacaagc tatcgaatta tttgatcacc aaggtgcaga gccagccttc ttattcggcc
     3001 ttgaattgat catatgcgga ttagaaaaac aacttaaatg tgaaagtggg tcctaataat
     3061 tggtaacgaa tcagacaatt gacggctcga gggagtagca tagggtttgc agaatccctg
     3121 cttcgtccat ttgacaggca cattatgcat cgatgataag ctgtcaaaca tgagcagatc
     3181 ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc
     3241 tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcaaa
     3301 tattttatct gaggtgcttc ctcgctcact gactcgctgc acgaggcaga cctcagcgct
     3361 agcggagtgt atactggctt actatgttgg cactgatgag ggtgtcagtg aagtgcttca
     3421 tgtggcagga gaaaaaaggc tgcaccggtg cgtcagcaga atatgtgata caggatatat
     3481 tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg
     3541 gcttacgaac ggggcggaga tttcccggaa gatgccagga agatacttaa cagggaagtg
     3601 agagggccgc ggcaaagccg tttttccata ggctccgccc ccctgacaag catcacgaaa
     3661 tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     3721 cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc ggtgtcattc
     3781 cgctgttatg gccgcgtttg tctcattcca cgcctgacac tcagttccgg gtaggcagtt
     3841 cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc
     3901 ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact ggcagcagcc
     3961 actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag gctaaactga
     4021 aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca aagagttggt
     4081 agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt cagagcaaga
     4141 gattacgcgc agaccaaaac gatctcaaga agatcatctt attaaggggt ctgacgctca
     4201 gtggaacgaa aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccttag
     4261 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     4321 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     4381 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     4441 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     4501 tccggtgaga atggcaaaag cttatgcatt tctttccaga cttgttcaac aggccagcca
     4561 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     4621 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     4681 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     4741 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     4801 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     4861 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     4921 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     4981 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctg
     5041 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     5101 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta catcagagat
     5161 tttgagacac aaccaattat tgaaggcctc cctaacgggg ggcctttttt tgtttctggt
     5221 ctcccgcttc tgtcttctgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     5281 aaaataccgc atcaggcgcc attagccatt caggctgcgc aactgttggg aagggcgatc
     5341 ggtgtgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     5401 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagt
//
