LOCUS       sequence-141103-        5705 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BM3R1-B2 gate.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5705)
  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 5705)
  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: pBM3R1-B2
FEATURES             Location/Qualifiers
     source          1..5705
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(308..852)
                     /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(388..407)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(941..1756)
                     /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(941..1756)
                     /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     1664..1683
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1748..1767
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     promoter        join(1878..1883,1884..1899,1900..1906)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    1912..1931
                     /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."
     misc_RNA        2756..2805
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(2855..2857,2858..2860,2861..3574)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2900..2921)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3161..3180)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3508..3529
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3722..4804
                     /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(3741..3760)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4823..5446
                     /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(4896..4915)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5629..5648)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gcttcctcgc tcactgactc gctgcacgag gcagacctca gcgctagcgg agtgtatact
       61 ggcttactat gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa
      121 aaggctgcac cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc
      181 actgactcgc tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc
      241 ggagatttcc tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa
      301 agccgttttt ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc
      361 agtggtggcg aaacccgaca ggactataaa gataccaggc gtttcccctg gcggctccct
      421 cgtgcgctct cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg
      481 tttgtctcat tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact
      541 gtatgcacga accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg
      601 agtccaaccc ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta
      661 gaggagttag tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt
      721 gactgcgctc ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc
      781 gaaaaaccgc cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca
      841 aaacgatctc aagaagatca tcttattaag gggtctgacg ctcagtggaa cgaaaaatca
      901 atctaaagta tatatgagta aacttggtct gacagttacc ttagaaaaac tcatcgagca
      961 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1021 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1081 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     1141 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     1201 aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     1261 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     1321 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     1381 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     1441 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     1501 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     1561 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     1621 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     1681 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc
     1741 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     1801 ttcatgatga tatattttta tcttgtgcaa tgtacatcag agattttgag acacaaggag
     1861 aacgatcgtt ggctgtgttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag
     1921 cgctcacaat tagactgtcg ccggatgtgt atccgacctg acgatggccc aaaagggccg
     1981 aaacagtcct ctacaaataa ttttgtttaa ctatggacta tgtttttcaa agacgaaaaa
     2041 ctactagatg gaaagcaccc cgaccaaaca gaaagcaatt tttagcgcaa gcctgctgct
     2101 gtttgcagaa cgtggttttg atgcaaccac catgccgatg attgcagaaa atgcaaaagt
     2161 tggtgcaggc accatttatc gctatttcaa aaacaaagaa agcctggtga acgaactgtt
     2221 tcagcagcat gttaatgaat ttctgcagtg tattgaaagc ggtctggcaa atgaacgtga
     2281 tggttatcgt gatggctttc atcacatttt tgaaggtatg gtgaccttta ccaaaaatca
     2341 tccgcgtgca ctgggtttta tcaaaaccca tagccagggc acctttctga ccgaagaaag
     2401 ccgtctggca tatcagaaac tggttgaatt tgtgtgcacc ttttttcgtg aaggtcagaa
     2461 acagggtgtg attcgtaatc tgccggaaaa tgcactgatt gcaattctgt ttggcagctt
     2521 tatggaagtg tatgaaatga tcgagaacga ttatctgagc ctgaccgatg aactgctgac
     2581 cggtgttgaa gaaagcctgt gggcagcact gagccgtcag agctaactcg gtaccaaatt
     2641 ccagaaaaga gacgctttcg agcgtctttt ttcgttttgg tcctactaat ccgcgtgata
     2701 ggtctgattc gttaccaatt gacggaatga acgttcattc cgataatgct agcagctgtc
     2761 accggatgtg ctttccggtc tgatgagtcc gtgaggacga aacagcctct acaaataatt
     2821 ttgtttaata ctagagaaag aggggaaata ctagatggtg agcaagggcg aggagctgtt
     2881 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
     2941 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
     3001 caccacaggc aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcct
     3061 gcaatgcttc gcccgctacc ccgaccacat gaagctgcac gacttcttca agtccgccat
     3121 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
     3181 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
     3241 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
     3301 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     3361 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acaccccaat
     3421 cggcgacggc cccgtgctgc tgcccgacaa ccactacctt agctaccagt ccgccctgag
     3481 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     3541 gatcactctc ggcatggacg agctgtacaa gtaactcggt accaaattcc agaaaagagg
     3601 cctcccgaaa ggggggcctt ttttcgtttt ggtccaatgg cggcgcgcca tcgaatggcg
     3661 caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca gggtggtgaa
     3721 tatgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt
     3781 ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc
     3841 ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg cgggcaaaca
     3901 gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt cgcaaattgt
     3961 cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt cgatggtaga
     4021 acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag
     4081 tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg aagctgcctg
     4141 cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca acagtattat
     4201 tttctcccat gaggacggta cgcgactggg cgtggagcat ctggtcgcat tgggtcacca
     4261 gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg
     4321 ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg aaggcgactg
     4381 gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca tcgttcccac
     4441 tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca ttaccgagtc
     4501 cgggctgcgc gttggtgcgg atatctcggt agtgggatac gacgataccg aagatagctc
     4561 atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag
     4621 cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc agctgttgcc
     4681 agtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg cctctccccg
     4741 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     4801 gtgataatcc aggaggaaaa aaatgtccag attagataaa agtaaagtga ttaacagcgc
     4861 attagagctg cttaatgagg tcggaatcga aggtttaaca acccgtaaac tcgcccagaa
     4921 gctaggtgta gagcagccta cattgtattg gcatgtaaaa aataagcggg ctttgctcga
     4981 cgccttagcc attgagatgt tagataggca ccatactcac ttttgccctt tagaagggga
     5041 aagctggcaa gattttttac gtaataacgc taaaagtttt agatgtgctt tactaagtca
     5101 tcgcgatgga gcaaaagtac atttaggtac acggcctaca gaaaaacagt atgaaactct
     5161 cgaaaatcaa ttagcctttt tatgccaaca aggtttttca ctagagaatg cattatatgc
     5221 actcagcgct gtggggcatt ttactttagg ttgcgtattg gaagatcaag agcatcaagt
     5281 cgctaaagaa gaaagggaaa cacctactac tgatagtatg ccgccattat tacgacaagc
     5341 tatcgaatta tttgatcacc aaggtgcaga gccagccttc ttattcggcc ttgaattgat
     5401 catatgcgga ttagaaaaac aacttaaatg tgaaagtggg tcctaataat tggtaacgaa
     5461 tcagacaatt gacggctcga gggagtagca tagggtttgc agaatccctg cttcgtccat
     5521 ttgacaggca cattatgcat cgatgataag ctgtcaaaca tgagcagatc ctctacgccg
     5581 gacgcatcgt ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg
     5641 acatcaccga tggggaagat cgggctcgcc acttcgggct catgagcaaa tattttatct
     5701 gaggt
//
