LOCUS       sequence-73330-e        7321 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7321)
  AUTHORS   Stanton BC, Nielsen AA, Tamsir A, Clancy K, Peterson T, Voigt CA
  TITLE     Genomic mining of prokaryotic repressors for orthogonal logic gates.
  JOURNAL   Nat Chem Biol. 2013 Dec 8. doi: 10.1038/nchembio.1411.
  PUBMED    24316737
REFERENCE   2  (bases 1 to 7321)
  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: "Nat Chem 
            Biol. 2013 Dec 8. doi: 10.1038/nchembio.1411."
COMMENT     Sequence Label: pRF-PhlF
FEATURES             Location/Qualifiers
     source          1..7321
                     /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(1877..2959)
                     /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     2921..2940
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(3094..3099,3100..3115,3116..3122)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    3128..3147
                     /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."
     terminator      3807..3878
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3894..3921
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             4002..4013
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(4020..4022,4023..4025,4026..4739)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4065..4086)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4326..4345)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4673..4694
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4779..4796)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      4949..5035
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      5127..5154
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        5173..5264
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5265..5333,5334..6125)
                     /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      6167..6622
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6254..6273)
                     /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 tcgagccgtc aattgtctga ttcgttacca
     1081 attattaatt tttaaagtat gggcaatcaa ttgctcctgt taaaattgct ttagaaatac
     1141 tttggcagcg gtttgttgta ttgagtttca tttgcgcatt ggttaaatgg aaagtgacag
     1201 tacgctcact gcaacctaat atttttgaaa tatcccaaga gctttttcct tcgcatgccc
     1261 acgctaaaca ttctttttct cttttggtta aatcgttgtt tgatttatta tttgctatat
     1321 ttatttttcg ataattatca actagagaag gaacaattaa tggtatgttc atacacgcat
     1381 gtaaaaataa actatctata tagttgtctt tttctgaatg tgcaaaacta agcattccga
     1441 agccattgtt agccgtatga atagggaaac taaacccagt gataagacct gatgttttcg
     1501 cttctttaat tacatttgga gattttttat ttacagcatt gttttcaaat atattccaat
     1561 taattggtga atgattggag ttagaataat ctactatagg atcatatttt attaaattag
     1621 cgtcatcata atattgcctc cattttttag ggtaattatc taggattgaa atatcagatt
     1681 taaccataga atgaggataa atgatcgcga gtaaataata ttcacaatgt accattttag
     1741 tcatatcaga taagcattga ttaatatcat tattgcttct acaagcttta attttattaa
     1801 ttattctgta tgtgtcgtcg gcatttatgt ttttcatcga ggcctttcct gtgtgacccg
     1861 ctggaatagg atcttatcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1921 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     1981 ttcttttcac cagtgagact ggcaacagct gattgccctt caccgcctgg ccctgagaga
     2041 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     2101 ttaacggcgg gatataacat gagctatctt cggtatcgtc gtatcccact accgagatat
     2161 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     2221 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     2281 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     2341 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     2401 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     2461 taccgtcctc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     2521 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     2581 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     2641 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     2701 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     2761 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     2821 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     2881 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     2941 ataacgttac tggtttcata ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     3001 ccataccgcg aaaggttttg cgccattcga tggcgcgccg cttcgtcagg ccacatagct
     3061 ttcttgttct gatcggaacg atcgttggct gtgttgacaa ttaatcatcg gctcgtataa
     3121 tgtgtggaat tgtgagcgct cacaattcta tggactatgt ttgaaaggga gaaatactag
     3181 atggcacgta ccccgagccg tagcagcatt ggtagcctgc gtagtccgca tacccataaa
     3241 gcaattctga ccagcaccat tgaaatcctg aaagaatgtg gttatagcgg tctgagcatt
     3301 gaaagcgttg cacgtcgtgc cggtgcaagc aaaccgacca tttatcgttg gtggaccaat
     3361 aaagcagcac tgattgccga agtgtatgaa aatgaaagcg aacaggtgcg taaatttccg
     3421 gatctgggta gctttaaagc cgatctggat tttctgctgc gtaatctgtg gaaagtttgg
     3481 cgtgaaacca tttgtggtga agcatttcgt tgtgttattg cagaagcaca gctggaccct
     3541 gcaaccctga cccagctgaa agatcagttt atggaacgtc gtcgtgagat gccgaaaaaa
     3601 ctggttgaaa atgccattag caatggtgaa ctgccgaaag ataccaatcg tgaactgctg
     3661 ctggatatga tttttggttt ttgttggtat cgcctgctga ccgaacagct gaccgttgaa
     3721 caggatattg aagaatttac cttcctgctg attaatggtg tttgtccggg tacacagcgt
     3781 taactagggc ccataccccc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg
     3841 cctttcgttt tatctgttgt ttgtcggtga acgctctcta ctagagtcac actggctcac
     3901 cttcgggtgg gcctttctgc gtttatacga cgtacggtgg aatctgattc gttaccaatt
     3961 gacatgatac gaaacgtacc gtatcgttaa ggttactaga gaaagaggag aaatactaga
     4021 tggtgagcaa gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg
     4081 gcgacgtaaa cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg
     4141 gcaagctgac cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc
     4201 tcgtgaccac cttcggctac ggcctgcaat gcttcgcccg ctaccccgac cacatgaagc
     4261 tgcacgactt cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct
     4321 tcaaggacga cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg
     4381 tgaaccgcat cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca
     4441 agctggagta caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg
     4501 gcatcaaggt gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg
     4561 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     4621 acctgagcta ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc
     4681 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagtaat
     4741 aagcttggct gttttggcgg atgagagaag attttcagcc tgatacagat taaatcagaa
     4801 cgcagaagcg gtctgataaa acagaatttg cctggcggca gtagcgcggt ggtcccacct
     4861 gaccccatgc cgaactcaga agtgaaacgc cgtagcgccg atggtagtgt ggggtctccc
     4921 catgcgagag tagggaactg ccaggcatca aataaaacga aaggctcagt cgaaagactg
     4981 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc ctgagtagga caaatccgcc
     5041 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
     5101 ataaactgcc aggcatcaaa ttaagcagaa ggccatcctg acggatggcc tttttgcgtt
     5161 tctacaaact cttttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5221 aataaccctg ataaatgctt caataatatt gaaaaaggaa gactatgagt attcaacatt
     5281 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5341 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5401 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5461 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     5521 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5581 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5641 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5701 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5761 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     5821 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5881 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5941 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6001 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6061 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6121 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttacgc gccctgtagc
     6181 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     6241 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     6301 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     6361 ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag
     6421 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     6481 acttgaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg
     6541 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     6601 aaaatattaa cgtttacaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     6661 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     6721 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     6781 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     6841 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     6901 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     6961 accagtcagg catttgagaa gcacacggtc acactgcttc cggtagtcaa taaaccggta
     7021 aaccagcaat agacataagc ggctatttaa cgaccctgcc ctgaaccgac gaccgggtcg
     7081 aatttgcttt cgaatttctg ccattcatcc gcttattatc acttattcag gcgtagcacc
     7141 aggcgtttaa gggcaccaat aactgcctta aaaaaattac gccccgccct gccactcatc
     7201 gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac agacggcatg
     7261 atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat atttgcccat
     7321 g
//
