LOCUS       sequence-106548-        7346 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Not gate genetic circuit containing four PhlF binding sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7346)
  TITLE     Salis Lab Plasmids
REFERENCE   2  (bases 1 to 7346)
  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"
COMMENT     Sequence Label: PhLF_N_4
FEATURES             Location/Qualifiers
     source          1..7346
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(132..149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(266..811)
                     /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(346..365)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     883..902
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(1836..2918)
                     /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     2880..2899
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(3053..3058,3059..3074,3075..3081)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3087..3106
                     /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        3109..3158
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     terminator      3832..3903
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3919..3946
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             4027..4038
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(4045..4047,4048..4050,4051..4764)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4090..4111)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4351..4370)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4698..4719
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4804..4821)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4974..5060
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5152..5179
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        5198..5289
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5290..5358,5359..6150)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      6192..6647
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6279..6298)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gctagcggag tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct
       61 tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata
      121 tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa
      181 atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa
      241 gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg
      301 aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt
      361 ttccccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca
      421 ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca
      481 gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta
      541 tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca
      601 gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac
      661 tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt
      721 ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca
      781 agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata
      841 tttgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
      901 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
      961 gatctgctca tgtttgacag cttatcatcg atgcataatg tgcctgtcaa atggacgaag
     1021 cagggattct gcaaacccta tgctactccc tcgagatgat acgaaacgta ccgtatcgtt
     1081 aaggttctag aatatatata tcatatatat atatatatga tacgaaacgt accgtatcgt
     1141 taaggtctct tcgctctcac tctcactcct catgatacga aacgtaccgt atcgttaagg
     1201 tgagctcata tgcatgccca cgctaaacat tctttttctc ttttggttaa atcgttgttt
     1261 gatttattat ttgctatatt tatttttcga taattatcaa ctagagaagg aacaattaat
     1321 ggtatgttca tacacgcatg taaaaataaa ctatctatat agttgtcttt ttctgaatgt
     1381 gcaaaactaa gcattccgaa gccattgtta gccgtatgaa tagggaaact aaacccagtg
     1441 ataagacctg atgttttcgc ttctttaatt acatttggag attttttatt tacagcattg
     1501 ttttcaaata tattccaatt aattggtgaa tgattggagt tagaataatc tactatagga
     1561 tcatatttta ttaaattagc gtcatcataa tattgcctcc attttttagg gtaattatct
     1621 aggattgaaa tatcagattt aaccatagaa tgaggataaa tgatcgcgag taaataatat
     1681 tcacaatgta ccattttagt catatcagat aagcattgat taatatcatt attgcttcta
     1741 caagctttaa ttttattaat tattctgtat gtgtcgtcgg catttatgtt tttcatcgag
     1801 gcctttcctg tgtgacccgc tggaatagga tcttatcact gcccgctttc cagtcgggaa
     1861 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     1921 ttgggcgcca gggtggtttt tcttttcacc agtgagactg gcaacagctg attgcccttc
     1981 accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga
     2041 aaatcctgtt tgatggtggt taacggcggg atataacatg agctatcttc ggtatcgtcg
     2101 tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt
     2161 gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc
     2221 agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct
     2281 atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc
     2341 gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga
     2401 tgctccacgc ccagtcgcgt accgtcctca tgggagaaaa taatactgtt gatgggtgtc
     2461 tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg
     2521 gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga
     2581 ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg
     2641 ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc
     2701 agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt
     2761 gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt
     2821 ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg
     2881 gcatactctg cgacatcgta taacgttact ggtttcatat tcaccaccct gaattgactc
     2941 tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat ggcgcgccgc
     3001 ttcgtcaggc cacatagctt tcttgttctg atcggaacga tcgttggctg tgttgacaat
     3061 taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc acaattagct gtcaccggat
     3121 gtgctttccg gtctgatgag tccgtgagga cgaaacagcc tctacaaata attttgttta
     3181 aattagtaac ataagcaaga gcctcatggc acgtaccccg agccgtagca gcattggtag
     3241 cctgcgtagt ccgcataccc ataaagcaat tctgaccagc accattgaaa tcctgaaaga
     3301 atgtggttat agcggtctga gcattgaaag cgttgcacgt cgtgccggtg caagcaaacc
     3361 gaccatttat cgttggtgga ccaataaagc agcactgatt gccgaagtgt atgaaaatga
     3421 aagcgaacag gtgcgtaaat ttccggatct gggtagcttt aaagccgatc tggattttct
     3481 gctgcgtaat ctgtggaaag tttggcgtga aaccatttgt ggtgaagcat ttcgttgtgt
     3541 tattgcagaa gcacagctgg accctgcaac cctgacccag ctgaaagatc agtttatgga
     3601 acgtcgtcgt gagatgccga aaaaactggt tgaaaatgcc attagcaatg gtgaactgcc
     3661 gaaagatacc aatcgtgaac tgctgctgga tatgattttt ggtttttgtt ggtatcgcct
     3721 gctgaccgaa cagctgaccg ttgaacagga tattgaagaa tttaccttcc tgctgattaa
     3781 tggtgtttgt ccgggtacac agcgttaact agggcccata cccccaggca tcaaataaaa
     3841 cgaaaggctc agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct
     3901 ctctactaga gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacgacgtac
     3961 ggtggaatct gattcgttac caattgacat gatacgaaac gtaccgtatc gttaaggtta
     4021 ctagagaaag aggagaaata ctagatggtg agcaagggcg aggagctgtt caccggggtg
     4081 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc
     4141 gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc
     4201 aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcct gcaatgcttc
     4261 gcccgctacc ccgaccacat gaagctgcac gacttcttca agtccgccat gcccgaaggc
     4321 tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag
     4381 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     4441 gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat
     4501 atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc
     4561 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     4621 cccgtgctgc tgcccgacaa ccactacctg agctaccagt ccgccctgag caaagacccc
     4681 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc
     4741 ggcatggacg agctgtacaa gtaataagct tggctgtttt ggcggatgag agaagatttt
     4801 cagcctgata cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg
     4861 cggcagtagc gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag
     4921 cgccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa
     4981 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     5041 ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg
     5101 gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca
     5161 tcctgacgga tggccttttt gcgtttctac aaactctttt gtttattttt ctaaatacat
     5221 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     5281 aggaagacta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     5341 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     5401 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     5461 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     5521 gtattatccc gtgttgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     5581 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     5641 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     5701 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     5761 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     5821 accacgatgc ctgcagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     5881 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     5941 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     6001 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     6061 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     6121 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     6181 tagattgatt tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     6241 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     6301 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     6361 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     6421 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     6481 ctttaatagt ggactcttgt tccaaacttg aacaacactc aaccctatct cgggctattc
     6541 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     6601 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttaaa aggatctagg
     6661 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     6721 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     6781 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     6841 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     6901 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     6961 catacctcgc tctgctaatc ctgttaccag tcaggcattt gagaagcaca cggtcacact
     7021 gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc
     7081 ctgccctgaa ccgacgaccg ggtcgaattt gctttcgaat ttctgccatt catccgctta
     7141 ttatcactta ttcaggcgta gcaccaggcg tttaagggca ccaataactg ccttaaaaaa
     7201 attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga
     7261 catggaagcc atcacagacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt
     7321 cgccttgcgt ataatatttg cccatg
//
