LOCUS       sequence-141110-        5721 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PhlF-P1 gate.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5721)
  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 5721)
  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-P1
FEATURES             Location/Qualifiers
     source          1..5721
                     /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        2772..2821
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     CDS             join(2871..2873,2874..2876,2877..3590)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2916..2937)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3177..3196)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3524..3545
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3738..4820
                     /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(3757..3776)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4839..5462
                     /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(4912..4931)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(5645..5664)
                     /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 gtttaactat ggactatgtt tgaaagggag aaatactaga
     2041 tggcacgtac cccgagccgt agcagcattg gtagcctgcg tagtccgcat acccataaag
     2101 caattctgac cagcaccatt gaaatcctga aagaatgtgg ttatagcggt ctgagcattg
     2161 aaagcgttgc acgtcgtgcc ggtgcaagca aaccgaccat ttatcgttgg tggaccaata
     2221 aagcagcact gattgccgaa gtgtatgaaa atgaaagcga acaggtgcgt aaatttccgg
     2281 atctgggtag ctttaaagcc gatctggatt ttctgctgcg taatctgtgg aaagtttggc
     2341 gtgaaaccat ttgtggtgaa gcatttcgtt gtgttattgc agaagcacag ctggaccctg
     2401 caaccctgac ccagctgaaa gatcagttta tggaacgtcg tcgtgagatg ccgaaaaaac
     2461 tggttgaaaa tgccattagc aatggtgaac tgccgaaaga taccaatcgt gaactgctgc
     2521 tggatatgat ttttggtttt tgttggtatc gcctgctgac cgaacagctg accgttgaac
     2581 aggatattga agaatttacc ttcctgctga ttaatggtgt ttgtccgggt acacagcgtt
     2641 aaggaaacac agaaaaaagc ccgcacctga cagtgcgggc tttttttttc gaccaaaggt
     2701 actcgacgta cggtggaatc tgattcgtta ccaattgaca tgatacgaaa cgtaccgtat
     2761 cgttaaggta gctgtcaccg gatgtgcttt ccggtctgat gagtccgtga ggacgaaaca
     2821 gcctctacaa ataattttgt ttaatactag agaaagaggg gaaatactag atggtgagca
     2881 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     2941 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     3001 ccctgaagtt catctgcacc acaggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     3061 ccttcggcta cggcctgcaa tgcttcgccc gctaccccga ccacatgaag ctgcacgact
     3121 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     3181 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     3241 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     3301 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     3361 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     3421 agcagaacac cccaatcggc gacggccccg tgctgctgcc cgacaaccac taccttagct
     3481 accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     3541 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa ctcggtacca
     3601 aattccagaa aagaggcctc ccgaaagggg ggcctttttt cgttttggtc caatggcggc
     3661 gcgccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg gaagagagtc
     3721 aattcagggt ggtgaatatg aaaccagtaa cgttatacga tgtcgcagag tatgccggtg
     3781 tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct gcgaaaacgc
     3841 gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc gtggcacaac
     3901 aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg gccctgcacg
     3961 cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg
     4021 tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc
     4081 tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag gatgccattg
     4141 ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct gaccagacac
     4201 ccatcaacag tattattttc tcccatgagg acggtacgcg actgggcgtg gagcatctgg
     4261 tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct gtctcggcgc
     4321 gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag ccgatagcgg
     4381 aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa atgctgaatg
     4441 agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg ggcgcaatgc
     4501 gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg
     4561 ataccgaaga tagctcatgt tatatcccgc cgttaaccac catcaaacag gattttcgcc
     4621 tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag gcggtgaagg
     4681 gcaatcagct gttgccagtc tcactggtga aaagaaaaac caccctggcg cccaatacgc
     4741 aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc
     4801 gactggaaag cgggcagtga taatccagga ggaaaaaaat gtccagatta gataaaagta
     4861 aagtgattaa cagcgcatta gagctgctta atgaggtcgg aatcgaaggt ttaacaaccc
     4921 gtaaactcgc ccagaagcta ggtgtagagc agcctacatt gtattggcat gtaaaaaata
     4981 agcgggcttt gctcgacgcc ttagccattg agatgttaga taggcaccat actcactttt
     5041 gccctttaga aggggaaagc tggcaagatt ttttacgtaa taacgctaaa agttttagat
     5101 gtgctttact aagtcatcgc gatggagcaa aagtacattt aggtacacgg cctacagaaa
     5161 aacagtatga aactctcgaa aatcaattag cctttttatg ccaacaaggt ttttcactag
     5221 agaatgcatt atatgcactc agcgctgtgg ggcattttac tttaggttgc gtattggaag
     5281 atcaagagca tcaagtcgct aaagaagaaa gggaaacacc tactactgat agtatgccgc
     5341 cattattacg acaagctatc gaattatttg atcaccaagg tgcagagcca gccttcttat
     5401 tcggccttga attgatcata tgcggattag aaaaacaact taaatgtgaa agtgggtcct
     5461 aataattggt aacgaatcag acaattgacg gctcgaggga gtagcatagg gtttgcagaa
     5521 tccctgcttc gtccatttga caggcacatt atgcatcgat gataagctgt caaacatgag
     5581 cagatcctct acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct
     5641 ggcgcctata tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg
     5701 agcaaatatt ttatctgagg t
//
