LOCUS       sequence-73326-e        7351 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 7351)
  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 7351)
  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-McbR
FEATURES             Location/Qualifiers
     source          1..7351
                     /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      749..820
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      836..863
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             941..952
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(959..961,962..964,965..1678)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1004..1025)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1265..1284)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1612..1633
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1718..1735)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1888..1974
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2066..2093
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2112..2203
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2204..2272,2273..3064)
                     /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      3106..3561
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3193..3212)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4392..4409)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4526..5071)
                     /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(4606..4625)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5143..5162
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(6137..7219)
                     /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     7181..7200
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc acaattctat
       61 ggactatgta ggagaaatac tagatggcag caagcgcaag cggtaaaagc aaaaccagtg
      121 ccggtgcaaa tcgtcgtcgt aatcgtccga gtccgcgtca gcgtctgctg gatagcgcaa
      181 ccaacctgtt taccaccgaa ggtattcgtg tgattggtat tgatcgtatt ctgcgtgaag
      241 cagatgttgc aaaagcaagc ctgtatagcc tgtttggtag caaagatgca ctggttattg
      301 cctatctgga aaatctggat cagctgtggc gtgaagcatg gcgtgaacgt accgttggta
      361 tgaaagatcc ggaagataaa atcattgcgt tctttgatca gtgcatcgaa gaagaaccgg
      421 aaaaagattt tcgcggtagc cattttcaga atgcagcaag cgaatatccg cgtccggaaa
      481 ccgatagcga aaaaggtatt gttgcagcag ttctggaaca tcgtgaatgg tgtcataaaa
      541 ccctgaccga tctgctgacc gaaaaaaatg gttatccggg tacaacccag gcaaatcagc
      601 tgctggtttt tctggatggt ggtctggcag gtagccgtct ggttcataac attagtccgc
      661 tggaaaccgc acgtgatctg gcacgtcagc tgctgagcgc accgcctgca gattatagca
      721 tttaactagg gcccataccc ccaggcatca aataaaacga aaggctcagt cgaaagactg
      781 ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc tactagagtc acactggctc
      841 accttcgggt gggcctttct gcgtttatac gacgtacggt ggaagattcg ttaccaattg
      901 acactagacc ggtctgtcta tataatgcta gctactagag aaagaggaga aatactagat
      961 ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     1021 cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg
     1081 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     1141 cgtgaccacc ttcggctacg gcctgcaatg cttcgcccgc taccccgacc acatgaagct
     1201 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt
     1261 caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     1321 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     1381 gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg
     1441 catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     1501 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     1561 cctgagctac cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     1621 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaata
     1681 agcttggctg ttttggcgga tgagagaaga ttttcagcct gatacagatt aaatcagaac
     1741 gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg
     1801 accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc
     1861 atgcgagagt agggaactgc caggcatcaa ataaaacgaa aggctcagtc gaaagactgg
     1921 gcctttcgtt ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccg
     1981 ggagcggatt tgaacgttgc gaagcaacgg cccggagggt ggcgggcagg acgcccgcca
     2041 taaactgcca ggcatcaaat taagcagaag gccatcctga cggatggcct ttttgcgttt
     2101 ctacaaactc ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca
     2161 ataaccctga taaatgcttc aataatattg aaaaaggaag actatgagta ttcaacattt
     2221 ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga
     2281 aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga
     2341 actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat
     2401 gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg acgccgggca
     2461 agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt
     2521 cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac
     2581 catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct
     2641 aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga
     2701 gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac
     2761 aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat
     2821 agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg
     2881 ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc
     2941 actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc
     3001 aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg
     3061 gtaactgtca gaccaagttt actcatatat actttagatt gatttacgcg ccctgtagcg
     3121 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     3181 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     3241 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     3301 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     3361 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     3421 cttgaacaac actcaaccct atctcgggct attcttttga tttataaggg attttgccga
     3481 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     3541 aaatattaac gtttacaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     3601 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     3661 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     3721 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     3781 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     3841 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     3901 ccagtcaggc atttgagaag cacacggtca cactgcttcc ggtagtcaat aaaccggtaa
     3961 accagcaata gacataagcg gctatttaac gaccctgccc tgaaccgacg accgggtcga
     4021 atttgctttc gaatttctgc cattcatccg cttattatca cttattcagg cgtagcacca
     4081 ggcgtttaag ggcaccaata actgccttaa aaaaattacg ccccgccctg ccactcatcg
     4141 cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca gacggcatga
     4201 tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg
     4261 gctagcggag tgtatactgg cttactatgt tggcactgat gagggtgtca gtgaagtgct
     4321 tcatgtggca ggagaaaaaa ggctgcaccg gtgcgtcagc agaatatgtg atacaggata
     4381 tattccgctt cctcgctcac tgactcgcta cgctcggtcg ttcgactgcg gcgagcggaa
     4441 atggcttacg aacggggcgg agatttcctg gaagatgcca ggaagatact taacagggaa
     4501 gtgagagggc cgcggcaaag ccgtttttcc ataggctccg cccccctgac aagcatcacg
     4561 aaatctgacg ctcaaatcag tggtggcgaa acccgacagg actataaaga taccaggcgt
     4621 ttccccctgg cggctccctc gtgcgctctc ctgttcctgc ctttcggttt accggtgtca
     4681 ttccgctgtt atggccgcgt ttgtctcatt ccacgcctga cactcagttc cgggtaggca
     4741 gttcgctcca agctggactg tatgcacgaa ccccccgttc agtccgaccg ctgcgcctta
     4801 tccggtaact atcgtcttga gtccaacccg gaaagacatg caaaagcacc actggcagca
     4861 gccactggta attgatttag aggagttagt cttgaagtca tgcgccggtt aaggctaaac
     4921 tgaaaggaca agttttggtg actgcgctcc tccaagccag ttacctcggt tcaaagagtt
     4981 ggtagctcag agaaccttcg aaaaaccgcc ctgcaaggcg gttttttcgt tttcagagca
     5041 agagattacg cgcagaccaa aacgatctca agaagatcat cttattaatc agataaaata
     5101 tttgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     5161 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     5221 gatctgctca tgtttgacag cttatcatcg atgcataatg tgcctgtcaa atggacgaag
     5281 cagggattct gcaaacccta tgctactccc tcgagccgtc aattgtctga ttcgttacca
     5341 attattaatt tttaaagtat gggcaatcaa ttgctcctgt taaaattgct ttagaaatac
     5401 tttggcagcg gtttgttgta ttgagtttca tttgcgcatt ggttaaatgg aaagtgacag
     5461 tacgctcact gcaacctaat atttttgaaa tatcccaaga gctttttcct tcgcatgccc
     5521 acgctaaaca ttctttttct cttttggtta aatcgttgtt tgatttatta tttgctatat
     5581 ttatttttcg ataattatca actagagaag gaacaattaa tggtatgttc atacacgcat
     5641 gtaaaaataa actatctata tagttgtctt tttctgaatg tgcaaaacta agcattccga
     5701 agccattgtt agccgtatga atagggaaac taaacccagt gataagacct gatgttttcg
     5761 cttctttaat tacatttgga gattttttat ttacagcatt gttttcaaat atattccaat
     5821 taattggtga atgattggag ttagaataat ctactatagg atcatatttt attaaattag
     5881 cgtcatcata atattgcctc cattttttag ggtaattatc taggattgaa atatcagatt
     5941 taaccataga atgaggataa atgatcgcga gtaaataata ttcacaatgt accattttag
     6001 tcatatcaga taagcattga ttaatatcat tattgcttct acaagcttta attttattaa
     6061 ttattctgta tgtgtcgtcg gcatttatgt ttttcatcga ggcctttcct gtgtgacccg
     6121 ctggaatagg atcttatcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     6181 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     6241 ttcttttcac cagtgagact ggcaacagct gattgccctt caccgcctgg ccctgagaga
     6301 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     6361 ttaacggcgg gatataacat gagctatctt cggtatcgtc gtatcccact accgagatat
     6421 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     6481 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     6541 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     6601 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     6661 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     6721 taccgtcctc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     6781 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     6841 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     6901 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     6961 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     7021 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     7081 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     7141 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     7201 ataacgttac tggtttcata ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     7261 ccataccgcg aaaggttttg cgccattcga tggcgcgccg cttcgtcagg ccacatagct
     7321 ttcttgttct gatcggaacg atcgttggct g
//
