LOCUS       sequence-73323-e        7298 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 7298)
  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 7298)
  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-LmrA
FEATURES             Location/Qualifiers
     source          1..7298
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(3..8,9..24,25..31)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..56
                     /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      681..752
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      768..795
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             888..899
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(906..908,909..911,912..1625)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(951..972)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1212..1231)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1559..1580
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1665..1682)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1835..1921
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2013..2040
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2059..2150
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2151..2219,2220..3011)
                     /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      3053..3508
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3140..3159)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4339..4356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4473..5018)
                     /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(4553..4572)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5090..5109
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(6084..7166)
                     /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     7128..7147
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc acaattctat
       61 ggactatgtt ttcacacagg aaaggcctcg atgagctatg gtgatagccg tgaaaaaatt
      121 ctgagcgcag caacccgtct gtttcagctg cagggttatt atggcaccgg tctgaatcag
      181 attatcaaag aaagcggtgc accgaaaggt agcctgtatt atcattttcc gggtggtaaa
      241 gaacagctgg caattgaagc agtgaacgaa atgaaagaat atatccgcca gaaaatcgcc
      301 gattgtatgg aagcatgtac cgatccggca gaaggtattc aggcatttct gaaagaactg
      361 agctgtcagt ttagctgtac cgaagatatt gaaggtctgc cggttggtct gctggcagca
      421 gaaaccagcc tgaaaagcga accgctgcgt gaagcatgtc atgaagcata taaagaatgg
      481 gccagcgtgt atgaagaaaa actgcgtcag accggttgta gcgaaagccg tgcaaaagaa
      541 gcaagcaccg ttgttaatgc aatgattgaa ggtggtattc tgctgagcct gaccgcaaaa
      601 aatagcacac cgctgctgca tattagcagc tgtattccgg atctgctgaa acgttaacta
      661 gggcccatac ccccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc
      721 gttttatctg ttgtttgtcg gtgaacgctc tctactagag tcacactggc tcaccttcgg
      781 gtgggccttt ctgcgtttat acgacgtacg gtggaatctg attcgttacc aattgacaac
      841 tggtggtcga atcaagataa tagaccagtc actatatttt actagagaaa gaggagaaat
      901 actagatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
      961 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     1021 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1081 ccaccctcgt gaccaccttc ggctacggcc tgcaatgctt cgcccgctac cccgaccaca
     1141 tgaagctgca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1201 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1261 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1321 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1381 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1441 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1501 accactacct gagctaccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     1561 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     1621 agtaataagc ttggctgttt tggcggatga gagaagattt tcagcctgat acagattaaa
     1681 tcagaacgca gaagcggtct gataaaacag aatttgcctg gcggcagtag cgcggtggtc
     1741 ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg
     1801 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     1861 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     1921 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     1981 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt
     2041 tgcgtttcta caaactcttt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     2101 tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagact atgagtattc
     2161 aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc
     2221 acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt
     2281 acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt
     2341 ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg
     2401 ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact
     2461 caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg
     2521 ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga
     2581 aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg
     2641 aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgcagcaa
     2701 tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac
     2761 aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc
     2821 cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca
     2881 ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga
     2941 gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta
     3001 agcattggta actgtcagac caagtttact catatatact ttagattgat ttacgcgccc
     3061 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3121 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3181 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3241 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
     3301 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3361 ttccaaactt gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt
     3421 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3481 tttaacaaaa tattaacgtt tacaatttaa aaggatctag gtgaagatcc tttttgataa
     3541 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga
     3601 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
     3661 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
     3721 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc
     3781 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
     3841 cctgttacca gtcaggcatt tgagaagcac acggtcacac tgcttccggt agtcaataaa
     3901 ccggtaaacc agcaatagac ataagcggct atttaacgac cctgccctga accgacgacc
     3961 gggtcgaatt tgctttcgaa tttctgccat tcatccgctt attatcactt attcaggcgt
     4021 agcaccaggc gtttaagggc accaataact gccttaaaaa aattacgccc cgccctgcca
     4081 ctcatcgcag tactgttgta attcattaag cattctgccg acatggaagc catcacagac
     4141 ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt
     4201 gcccatggct agcggagtgt atactggctt actatgttgg cactgatgag ggtgtcagtg
     4261 aagtgcttca tgtggcagga gaaaaaaggc tgcaccggtg cgtcagcaga atatgtgata
     4321 caggatatat tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg
     4381 agcggaaatg gcttacgaac ggggcggaga tttcctggaa gatgccagga agatacttaa
     4441 cagggaagtg agagggccgc ggcaaagccg tttttccata ggctccgccc ccctgacaag
     4501 catcacgaaa tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac
     4561 caggcgtttc cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc
     4621 ggtgtcattc cgctgttatg gccgcgtttg tctcattcca cgcctgacac tcagttccgg
     4681 gtaggcagtt cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg
     4741 cgccttatcc ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact
     4801 ggcagcagcc actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag
     4861 gctaaactga aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca
     4921 aagagttggt agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt
     4981 cagagcaaga gattacgcgc agaccaaaac gatctcaaga agatcatctt attaatcaga
     5041 taaaatattt gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
     5101 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
     5161 cgtagaggat ctgctcatgt ttgacagctt atcatcgatg cataatgtgc ctgtcaaatg
     5221 gacgaagcag ggattctgca aaccctatgc tactccctcg agccgtcaat tgtctgattc
     5281 gttaccaatt attaattttt aaagtatggg caatcaattg ctcctgttaa aattgcttta
     5341 gaaatacttt ggcagcggtt tgttgtattg agtttcattt gcgcattggt taaatggaaa
     5401 gtgacagtac gctcactgca acctaatatt tttgaaatat cccaagagct ttttccttcg
     5461 catgcccacg ctaaacattc tttttctctt ttggttaaat cgttgtttga tttattattt
     5521 gctatattta tttttcgata attatcaact agagaaggaa caattaatgg tatgttcata
     5581 cacgcatgta aaaataaact atctatatag ttgtcttttt ctgaatgtgc aaaactaagc
     5641 attccgaagc cattgttagc cgtatgaata gggaaactaa acccagtgat aagacctgat
     5701 gttttcgctt ctttaattac atttggagat tttttattta cagcattgtt ttcaaatata
     5761 ttccaattaa ttggtgaatg attggagtta gaataatcta ctataggatc atattttatt
     5821 aaattagcgt catcataata ttgcctccat tttttagggt aattatctag gattgaaata
     5881 tcagatttaa ccatagaatg aggataaatg atcgcgagta aataatattc acaatgtacc
     5941 attttagtca tatcagataa gcattgatta atatcattat tgcttctaca agctttaatt
     6001 ttattaatta ttctgtatgt gtcgtcggca tttatgtttt tcatcgaggc ctttcctgtg
     6061 tgacccgctg gaataggatc ttatcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6121 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     6181 gtggtttttc ttttcaccag tgagactggc aacagctgat tgcccttcac cgcctggccc
     6241 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     6301 atggtggtta acggcgggat ataacatgag ctatcttcgg tatcgtcgta tcccactacc
     6361 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     6421 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     6481 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     6541 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     6601 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     6661 agtcgcgtac cgtcctcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     6721 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     6781 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     6841 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     6901 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     6961 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     7021 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     7081 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     7141 acatcgtata acgttactgg tttcatattc accaccctga attgactctc ttccgggcgc
     7201 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg cgcgccgctt cgtcaggcca
     7261 catagctttc ttgttctgat cggaacgatc gttggctg
//
