LOCUS       sequence-106547-        7232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Not gate genetic circuit containing two PhlF binding sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7232)
  TITLE     Salis Lab Plasmids
REFERENCE   2  (bases 1 to 7232)
  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_2
FEATURES             Location/Qualifiers
     source          1..7232
                     /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(1722..2804)
                     /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     2766..2785
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(2939..2944,2945..2960,2961..2967)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2973..2992
                     /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        2995..3044
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     terminator      3718..3789
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3805..3832
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             3913..3924
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(3931..3933,3934..3936,3937..4650)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3976..3997)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4237..4256)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4584..4605
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4690..4707)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4860..4946
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5038..5065
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        5084..5175
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5176..5244,5245..6036)
                     /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      6078..6533
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6165..6184)
                     /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 aacacacgca tgcccacgct aaacattctt tttctctttt ggttaaatcg
     1141 ttgtttgatt tattatttgc tatatttatt tttcgataat tatcaactag agaaggaaca
     1201 attaatggta tgttcataca cgcatgtaaa aataaactat ctatatagtt gtctttttct
     1261 gaatgtgcaa aactaagcat tccgaagcca ttgttagccg tatgaatagg gaaactaaac
     1321 ccagtgataa gacctgatgt tttcgcttct ttaattacat ttggagattt tttatttaca
     1381 gcattgtttt caaatatatt ccaattaatt ggtgaatgat tggagttaga ataatctact
     1441 ataggatcat attttattaa attagcgtca tcataatatt gcctccattt tttagggtaa
     1501 ttatctagga ttgaaatatc agatttaacc atagaatgag gataaatgat cgcgagtaaa
     1561 taatattcac aatgtaccat tttagtcata tcagataagc attgattaat atcattattg
     1621 cttctacaag ctttaatttt attaattatt ctgtatgtgt cgtcggcatt tatgtttttc
     1681 atcgaggcct ttcctgtgtg acccgctgga ataggatctt atcactgccc gctttccagt
     1741 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     1801 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agactggcaa cagctgattg
     1861 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     1921 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct atcttcggta
     1981 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     2041 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     2101 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     2161 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     2221 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     2281 accagatgct ccacgcccag tcgcgtaccg tcctcatggg agaaaataat actgttgatg
     2341 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     2401 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     2461 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     2521 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     2581 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     2641 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     2701 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     2761 acaccggcat actctgcgac atcgtataac gttactggtt tcatattcac caccctgaat
     2821 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggcg
     2881 cgccgcttcg tcaggccaca tagctttctt gttctgatcg gaacgatcgt tggctgtgtt
     2941 gacaattaat catcggctcg tataatgtgt ggaattgtga gcgctcacaa ttagctgtca
     3001 ccggatgtgc tttccggtct gatgagtccg tgaggacgaa acagcctcta caaataattt
     3061 tgtttaaatt agtaacataa gcaagagcct catggcacgt accccgagcc gtagcagcat
     3121 tggtagcctg cgtagtccgc atacccataa agcaattctg accagcacca ttgaaatcct
     3181 gaaagaatgt ggttatagcg gtctgagcat tgaaagcgtt gcacgtcgtg ccggtgcaag
     3241 caaaccgacc atttatcgtt ggtggaccaa taaagcagca ctgattgccg aagtgtatga
     3301 aaatgaaagc gaacaggtgc gtaaatttcc ggatctgggt agctttaaag ccgatctgga
     3361 ttttctgctg cgtaatctgt ggaaagtttg gcgtgaaacc atttgtggtg aagcatttcg
     3421 ttgtgttatt gcagaagcac agctggaccc tgcaaccctg acccagctga aagatcagtt
     3481 tatggaacgt cgtcgtgaga tgccgaaaaa actggttgaa aatgccatta gcaatggtga
     3541 actgccgaaa gataccaatc gtgaactgct gctggatatg atttttggtt tttgttggta
     3601 tcgcctgctg accgaacagc tgaccgttga acaggatatt gaagaattta ccttcctgct
     3661 gattaatggt gtttgtccgg gtacacagcg ttaactaggg cccatacccc caggcatcaa
     3721 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     3781 aacgctctct actagagtca cactggctca ccttcgggtg ggcctttctg cgtttatacg
     3841 acgtacggtg gaatctgatt cgttaccaat tgacatgata cgaaacgtac cgtatcgtta
     3901 aggttactag agaaagagga gaaatactag atggtgagca agggcgagga gctgttcacc
     3961 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     4021 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     4081 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta cggcctgcaa
     4141 tgcttcgccc gctaccccga ccacatgaag ctgcacgact tcttcaagtc cgccatgccc
     4201 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     4261 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     4321 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     4381 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     4441 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     4501 gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgc cctgagcaaa
     4561 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     4621 actctcggca tggacgagct gtacaagtaa taagcttggc tgttttggcg gatgagagaa
     4681 gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt
     4741 gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg
     4801 ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc
     4861 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     4921 tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac
     4981 ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga
     5041 aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttttgttt atttttctaa
     5101 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5161 tgaaaaagga agactatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5221 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5281 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5341 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5401 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     5461 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5521 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5581 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     5641 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     5701 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5761 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5821 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5881 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5941 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6001 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6061 atactttaga ttgatttacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     6121 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     6181 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     6241 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     6301 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     6361 cacgttcttt aatagtggac tcttgttcca aacttgaaca acactcaacc ctatctcggg
     6421 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     6481 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa tttaaaagga
     6541 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6601 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6661 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6721 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6781 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6841 cgcctacata cctcgctctg ctaatcctgt taccagtcag gcatttgaga agcacacggt
     6901 cacactgctt ccggtagtca ataaaccggt aaaccagcaa tagacataag cggctattta
     6961 acgaccctgc cctgaaccga cgaccgggtc gaatttgctt tcgaatttct gccattcatc
     7021 cgcttattat cacttattca ggcgtagcac caggcgttta agggcaccaa taactgcctt
     7081 aaaaaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc
     7141 tgccgacatg gaagccatca cagacggcat gatgaacctg aatcgccagc ggcatcagca
     7201 ccttgtcgcc ttgcgtataa tatttgccca tg
//
