LOCUS       sequence-141198-        5810 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SrpR-S4 gate.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5810)
  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 5810)
  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: pSrpR-S4
FEATURES             Location/Qualifiers
     source          1..5810
                     /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        2861..2910
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(2960..2962,2963..2965,2966..3679)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3005..3026)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3266..3285)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3613..3634
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3827..4909
                     /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(3846..3865)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4928..5551
                     /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(5001..5020)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5734..5753)
                     /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 tagcgctcaa cgggtgtgct tcccgttctg atgagtccgt gaggacgaaa
     1981 gcgcctctac aaataatttt gtttaactat ggactatgtt ttcacacagg aaataccagg
     2041 atggcacgta aaaccgcagc agaagcagaa gaaacccgtc agcgtattat tgatgcagca
     2101 ctggaagttt ttgttgcaca gggtgttagt gatgcaaccc tggatcagat tgcacgtaaa
     2161 gccggtgtta cccgtggtgc agtttattgg cattttaatg gtaaactgga agttctgcag
     2221 gcagttctgg caagccgtca gcatccgctg gaactggatt ttacaccgga tctgggtatt
     2281 gaacgtagct gggaagcagt tgttgttgca atgctggatg cagttcatag tccgcagagc
     2341 aaacagttta gcgaaattct gatttatcag ggtctggatg aaagcggtct gattcataat
     2401 cgtatggttc aggcaagcga tcgttttctg cagtatattc atcaggttct gcgtcatgca
     2461 gttacccagg gtgaactgcc gattaatctg gatctgcaga ccagcattgg tgtttttaaa
     2521 ggtctgatta ccggtctgct gtatgaaggt ctgcgtagca aagatcagca ggcacagatt
     2581 atcaaagttg cactgggtag cttttgggca ctgctgcgtg aaccgcctcg ttttctgctg
     2641 tgtgaagaag cacagattaa acaggtgaaa tccttcgaat aattcagcca aaaaacttaa
     2701 gaccgccggt cttgtccact accttgcagt aatgcggtgg acaggatcgg cggttttctt
     2761 ttctcttctc aatacttcta tgattggtcc agattcgtta ccaattgaca gctagctcag
     2821 tcctaggtat atacatacat gcttgtttgt ttgtaaacag ctgtcaccgg atgtgctttc
     2881 cggtctgatg agtccgtgag gacgaaacag cctctacaaa taattttgtt taatactaga
     2941 gaaagagggg aaatactaga tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc
     3001 catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg
     3061 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca caggcaagct
     3121 gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgcaat gcttcgcccg
     3181 ctaccccgac cacatgaagc tgcacgactt cttcaagtcc gccatgcccg aaggctacgt
     3241 ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg ccgaggtgaa
     3301 gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact tcaaggagga
     3361 cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg tctatatcat
     3421 ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     3481 cggcagcgtg cagctcgccg accactacca gcagaacacc ccaatcggcg acggccccgt
     3541 gctgctgccc gacaaccact accttagcta ccagtccgcc ctgagcaaag accccaacga
     3601 gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca ctctcggcat
     3661 ggacgagctg tacaagtaac tcggtaccaa attccagaaa agaggcctcc cgaaaggggg
     3721 gccttttttc gttttggtcc aatggcggcg cgccatcgaa tggcgcaaaa cctttcgcgg
     3781 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatatga aaccagtaac
     3841 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     3901 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     3961 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     4021 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     4081 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     4141 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     4201 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     4261 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgagga
     4321 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     4381 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     4441 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     4501 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     4561 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     4621 tgcggatatc tcggtagtgg gatacgacga taccgaagat agctcatgtt atatcccgcc
     4681 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     4741 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgccagtct cactggtgaa
     4801 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     4861 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgat aatccaggag
     4921 gaaaaaaatg tccagattag ataaaagtaa agtgattaac agcgcattag agctgcttaa
     4981 tgaggtcgga atcgaaggtt taacaacccg taaactcgcc cagaagctag gtgtagagca
     5041 gcctacattg tattggcatg taaaaaataa gcgggctttg ctcgacgcct tagccattga
     5101 gatgttagat aggcaccata ctcacttttg ccctttagaa ggggaaagct ggcaagattt
     5161 tttacgtaat aacgctaaaa gttttagatg tgctttacta agtcatcgcg atggagcaaa
     5221 agtacattta ggtacacggc ctacagaaaa acagtatgaa actctcgaaa atcaattagc
     5281 ctttttatgc caacaaggtt tttcactaga gaatgcatta tatgcactca gcgctgtggg
     5341 gcattttact ttaggttgcg tattggaaga tcaagagcat caagtcgcta aagaagaaag
     5401 ggaaacacct actactgata gtatgccgcc attattacga caagctatcg aattatttga
     5461 tcaccaaggt gcagagccag ccttcttatt cggccttgaa ttgatcatat gcggattaga
     5521 aaaacaactt aaatgtgaaa gtgggtccta ataattggta acgaatcaga caattgacgg
     5581 ctcgagggag tagcataggg tttgcagaat ccctgcttcg tccatttgac aggcacatta
     5641 tgcatcgatg ataagctgtc aaacatgagc agatcctcta cgccggacgc atcgtggccg
     5701 gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc accgatgggg
     5761 aagatcgggc tcgccacttc gggctcatga gcaaatattt tatctgaggt
//
