LOCUS       sequence-141111-        5725 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PhlF-P2 gate.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5725)
  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 5725)
  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: pPhlF-P2
FEATURES             Location/Qualifiers
     source          1..5725
                     /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        2776..2825
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(2875..2877,2878..2880,2881..3594)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2920..2941)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3181..3200)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3528..3549
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3742..4824
                     /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(3761..3780)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4843..5466
                     /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(4916..4935)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5649..5668)
                     /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 tagcggtcaa cgcatgtgct ttgcgttctg atgagacagt gatgtcgaaa
     1981 ccgcctctac aaataatttt gtttaaggag ctatggacta tgtttgaaag gctgaaatac
     2041 tagatggcac gtaccccgag ccgtagcagc attggtagcc tgcgtagtcc gcatacccat
     2101 aaagcaattc tgaccagcac cattgaaatc ctgaaagaat gtggttatag cggtctgagc
     2161 attgaaagcg ttgcacgtcg tgccggtgca agcaaaccga ccatttatcg ttggtggacc
     2221 aataaagcag cactgattgc cgaagtgtat gaaaatgaaa gcgaacaggt gcgtaaattt
     2281 ccggatctgg gtagctttaa agccgatctg gattttctgc tgcgtaatct gtggaaagtt
     2341 tggcgtgaaa ccatttgtgg tgaagcattt cgttgtgtta ttgcagaagc acagctggac
     2401 cctgcaaccc tgacccagct gaaagatcag tttatggaac gtcgtcgtga gatgccgaaa
     2461 aaactggttg aaaatgccat tagcaatggt gaactgccga aagataccaa tcgtgaactg
     2521 ctgctggata tgatttttgg tttttgttgg tatcgcctgc tgaccgaaca gctgaccgtt
     2581 gaacaggata ttgaagaatt taccttcctg ctgattaatg gtgtttgtcc gggtacacag
     2641 cgttaaggaa acacagaaaa aagcccgcac ctgacagtgc gggctttttt tttcgaccaa
     2701 aggtactcga cgtacggtgg aatctgattc gttaccaatt gacatgatac gaaacgtacc
     2761 gtatcgttaa ggtagctgtc accggatgtg ctttccggtc tgatgagtcc gtgaggacga
     2821 aacagcctct acaaataatt ttgtttaata ctagagaaag aggggaaata ctagatggtg
     2881 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     2941 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     3001 ctgaccctga agttcatctg caccacaggc aagctgcccg tgccctggcc caccctcgtg
     3061 accaccttcg gctacggcct gcaatgcttc gcccgctacc ccgaccacat gaagctgcac
     3121 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     3181 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     3241 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     3301 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     3361 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     3421 taccagcaga acaccccaat cggcgacggc cccgtgctgc tgcccgacaa ccactacctt
     3481 agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     3541 gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaactcggt
     3601 accaaattcc agaaaagagg cctcccgaaa ggggggcctt ttttcgtttt ggtccaatgg
     3661 cggcgcgcca tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag
     3721 agtcaattca gggtggtgaa tatgaaacca gtaacgttat acgatgtcgc agagtatgcc
     3781 ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa
     3841 acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca
     3901 caacaactgg cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg
     3961 cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc
     4021 gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat
     4081 cttctcgcgc aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc
     4141 attgctgtgg aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag
     4201 acacccatca acagtattat tttctcccat gaggacggta cgcgactggg cgtggagcat
     4261 ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg
     4321 gcgcgtctgc gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata
     4381 gcggaacggg aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg
     4441 aatgagggca tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca
     4501 atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg atatctcggt agtgggatac
     4561 gacgataccg aagatagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt
     4621 cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg
     4681 aagggcaatc agctgttgcc agtctcactg gtgaaaagaa aaaccaccct ggcgcccaat
     4741 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
     4801 tcccgactgg aaagcgggca gtgataatcc aggaggaaaa aaatgtccag attagataaa
     4861 agtaaagtga ttaacagcgc attagagctg cttaatgagg tcggaatcga aggtttaaca
     4921 acccgtaaac tcgcccagaa gctaggtgta gagcagccta cattgtattg gcatgtaaaa
     4981 aataagcggg ctttgctcga cgccttagcc attgagatgt tagataggca ccatactcac
     5041 ttttgccctt tagaagggga aagctggcaa gattttttac gtaataacgc taaaagtttt
     5101 agatgtgctt tactaagtca tcgcgatgga gcaaaagtac atttaggtac acggcctaca
     5161 gaaaaacagt atgaaactct cgaaaatcaa ttagcctttt tatgccaaca aggtttttca
     5221 ctagagaatg cattatatgc actcagcgct gtggggcatt ttactttagg ttgcgtattg
     5281 gaagatcaag agcatcaagt cgctaaagaa gaaagggaaa cacctactac tgatagtatg
     5341 ccgccattat tacgacaagc tatcgaatta tttgatcacc aaggtgcaga gccagccttc
     5401 ttattcggcc ttgaattgat catatgcgga ttagaaaaac aacttaaatg tgaaagtggg
     5461 tcctaataat tggtaacgaa tcagacaatt gacggctcga gggagtagca tagggtttgc
     5521 agaatccctg cttcgtccat ttgacaggca cattatgcat cgatgataag ctgtcaaaca
     5581 tgagcagatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt
     5641 tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct
     5701 catgagcaaa tattttatct gaggt
//
