LOCUS       sequence-141102-        5693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BM3R1-B1 gate.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5693)
  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 5693)
  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-B1
FEATURES             Location/Qualifiers
     source          1..5693
                     /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        2744..2793
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(2843..2845,2846..2848,2849..3562)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2888..2909)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3149..3168)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3496..3517
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3710..4792
                     /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(3729..3748)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4811..5434
                     /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(4884..4903)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5617..5636)
                     /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 tgttttaact actagatgga
     2041 aagcaccccg accaaacaga aagcaatttt tagcgcaagc ctgctgctgt ttgcagaacg
     2101 tggttttgat gcaaccacca tgccgatgat tgcagaaaat gcaaaagttg gtgcaggcac
     2161 catttatcgc tatttcaaaa acaaagaaag cctggtgaac gaactgtttc agcagcatgt
     2221 taatgaattt ctgcagtgta ttgaaagcgg tctggcaaat gaacgtgatg gttatcgtga
     2281 tggctttcat cacatttttg aaggtatggt gacctttacc aaaaatcatc cgcgtgcact
     2341 gggttttatc aaaacccata gccagggcac ctttctgacc gaagaaagcc gtctggcata
     2401 tcagaaactg gttgaatttg tgtgcacctt ttttcgtgaa ggtcagaaac agggtgtgat
     2461 tcgtaatctg ccggaaaatg cactgattgc aattctgttt ggcagcttta tggaagtgta
     2521 tgaaatgatc gagaacgatt atctgagcct gaccgatgaa ctgctgaccg gtgttgaaga
     2581 aagcctgtgg gcagcactga gccgtcagag ctaactcggt accaaattcc agaaaagaga
     2641 cgctttcgag cgtctttttt cgttttggtc ctactaatcc gcgtgatagg tctgattcgt
     2701 taccaattga cggaatgaac gttcattccg ataatgctag cagctgtcac cggatgtgct
     2761 ttccggtctg atgagtccgt gaggacgaaa cagcctctac aaataatttt gtttaatact
     2821 agagaaagag gggaaatact agatggtgag caagggcgag gagctgttca ccggggtggt
     2881 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     2941 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccacaggcaa
     3001 gctgcccgtg ccctggccca ccctcgtgac caccttcggc tacggcctgc aatgcttcgc
     3061 ccgctacccc gaccacatga agctgcacga cttcttcaag tccgccatgc ccgaaggcta
     3121 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     3181 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     3241 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
     3301 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
     3361 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac accccaatcg gcgacggccc
     3421 cgtgctgctg cccgacaacc actaccttag ctaccagtcc gccctgagca aagaccccaa
     3481 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     3541 catggacgag ctgtacaagt aactcggtac caaattccag aaaagaggcc tcccgaaagg
     3601 ggggcctttt ttcgttttgg tccaatggcg gcgcgccatc gaatggcgca aaacctttcg
     3661 cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaata tgaaaccagt
     3721 aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt
     3781 gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga
     3841 gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat
     3901 tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa
     3961 atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt
     4021 cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat
     4081 taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc
     4141 ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga
     4201 ggacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct
     4261 gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata
     4321 tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc
     4381 cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt
     4441 tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt
     4501 tggtgcggat atctcggtag tgggatacga cgataccgaa gatagctcat gttatatccc
     4561 gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt
     4621 gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgccag tctcactggt
     4681 gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     4741 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gataatccag
     4801 gaggaaaaaa atgtccagat tagataaaag taaagtgatt aacagcgcat tagagctgct
     4861 taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagc taggtgtaga
     4921 gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacg ccttagccat
     4981 tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaa gctggcaaga
     5041 ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatc gcgatggagc
     5101 aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcg aaaatcaatt
     5161 agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcac tcagcgctgt
     5221 ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcg ctaaagaaga
     5281 aagggaaaca cctactactg atagtatgcc gccattatta cgacaagcta tcgaattatt
     5341 tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatca tatgcggatt
     5401 agaaaaacaa cttaaatgtg aaagtgggtc ctaataattg gtaacgaatc agacaattga
     5461 cggctcgagg gagtagcata gggtttgcag aatccctgct tcgtccattt gacaggcaca
     5521 ttatgcatcg atgataagct gtcaaacatg agcagatcct ctacgccgga cgcatcgtgg
     5581 ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg
     5641 gggaagatcg ggctcgccac ttcgggctca tgagcaaata ttttatctga ggt
//
