LOCUS       sequence-75151-e        7690 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 7690)
  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 7690)
  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-Orf2
FEATURES             Location/Qualifiers
     source          1..7690
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(40..57)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      210..296
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      388..415
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        434..525
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(526..594,595..1386)
                     /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      1428..1883
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1515..1534)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(2714..2731)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2848..3393)
                     /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(2928..2947)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3465..3484
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(4459..5541)
                     /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     5503..5522
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5676..5681,5682..5697,5698..5704)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    5710..5729
                     /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."
     RBS             5747..5758
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     terminator      6658..6729
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      6745..6772
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             6867..6878
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     RBS             6953..6964
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(6971..6973,6974..6976,6977..7690)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(7016..7037)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(7277..7296)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7624..7645
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 taagcttggc tgttttggcg gatgagagaa gattttcagc ctgatacaga ttaaatcaga
       61 acgcagaagc ggtctgataa aacagaattt gcctggcggc agtagcgcgg tggtcccacc
      121 tgaccccatg ccgaactcag aagtgaaacg ccgtagcgcc gatggtagtg tggggtctcc
      181 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact
      241 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
      301 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc
      361 cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt
      421 ttctacaaac tcttttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
      481 caataaccct gataaatgct tcaataatat tgaaaaagga agactatgag tattcaacat
      541 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
      601 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
      661 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
      721 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
      781 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
      841 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
      901 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
      961 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     1021 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     1081 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     1141 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     1201 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     1261 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     1321 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     1381 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttacg cgccctgtag
     1441 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1501 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     1561 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     1621 cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata
     1681 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     1741 aacttgaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc
     1801 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     1861 caaaatatta acgtttacaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     1921 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     1981 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2041 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2101 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     2161 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2221 taccagtcag gcatttgaga agcacacggt cacactgctt ccggtagtca ataaaccggt
     2281 aaaccagcaa tagacataag cggctattta acgaccctgc cctgaaccga cgaccgggtc
     2341 gaatttgctt tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcac
     2401 caggcgttta agggcaccaa taactgcctt aaaaaaatta cgccccgccc tgccactcat
     2461 cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca cagacggcat
     2521 gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca
     2581 tggctagcgg agtgtatact ggcttactat gttggcactg atgagggtgt cagtgaagtg
     2641 cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca gcagaatatg tgatacagga
     2701 tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg cggcgagcgg
     2761 aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata cttaacaggg
     2821 aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg acaagcatca
     2881 cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa gataccaggc
     2941 gtttccccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt
     3001 cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg
     3061 cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct
     3121 tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag
     3181 cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa
     3241 actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag
     3301 ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag
     3361 caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa tcagataaaa
     3421 tatttgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     3481 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     3541 aggatctgct catgtttgac agcttatcat cgatgcataa tgtgcctgtc aaatggacga
     3601 agcagggatt ctgcaaaccc tatgctactc cctcgagccg tcaattgtct gattcgttac
     3661 caattattaa tttttaaagt atgggcaatc aattgctcct gttaaaattg ctttagaaat
     3721 actttggcag cggtttgttg tattgagttt catttgcgca ttggttaaat ggaaagtgac
     3781 agtacgctca ctgcaaccta atatttttga aatatcccaa gagctttttc cttcgcatgc
     3841 ccacgctaaa cattcttttt ctcttttggt taaatcgttg tttgatttat tatttgctat
     3901 atttattttt cgataattat caactagaga aggaacaatt aatggtatgt tcatacacgc
     3961 atgtaaaaat aaactatcta tatagttgtc tttttctgaa tgtgcaaaac taagcattcc
     4021 gaagccattg ttagccgtat gaatagggaa actaaaccca gtgataagac ctgatgtttt
     4081 cgcttcttta attacatttg gagatttttt atttacagca ttgttttcaa atatattcca
     4141 attaattggt gaatgattgg agttagaata atctactata ggatcatatt ttattaaatt
     4201 agcgtcatca taatattgcc tccatttttt agggtaatta tctaggattg aaatatcaga
     4261 tttaaccata gaatgaggat aaatgatcgc gagtaaataa tattcacaat gtaccatttt
     4321 agtcatatca gataagcatt gattaatatc attattgctt ctacaagctt taattttatt
     4381 aattattctg tatgtgtcgt cggcatttat gtttttcatc gaggcctttc ctgtgtgacc
     4441 cgctggaata ggatcttatc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     4501 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt
     4561 ttttcttttc accagtgaga ctggcaacag ctgattgccc ttcaccgcct ggccctgaga
     4621 gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt
     4681 ggttaacggc gggatataac atgagctatc ttcggtatcg tcgtatccca ctaccgagat
     4741 atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg
     4801 atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg
     4861 ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt
     4921 gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg
     4981 gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg
     5041 cgtaccgtcc tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag
     5101 aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag
     5161 cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt
     5221 acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc
     5281 ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt
     5341 ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat
     5401 gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct
     5461 ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc
     5521 gtataacgtt actggtttca tattcaccac cctgaattga ctctcttccg ggcgctatca
     5581 tgccataccg cgaaaggttt tgcgccattc gatggcgcgc cgcttcgtca ggccacatag
     5641 ctttcttgtt ctgatcggaa cgatcgttgg ctgtgttgac aattaatcat cggctcgtat
     5701 aatgtgtgga attgtgagcg ctcacaattc tatggactat gttttgaaag aggagaaaca
     5761 ctagatgcag cagcagcatg aagttgcagc acgtgttcgt cgtgttattg atgcagccgg
     5821 tgttagcgca cgtgaatttg cacgtcgtat tgttattgat cgtcgtagca gtccgggtcc
     5881 gagcaccgca ccgggtgcaa gtccgcctcc gaattggcct ggtagccgta caccggcagc
     5941 atggacctgg gatggttgta gcggtctgcg tccggttcgt cgtcgtcgta gcaccgttcg
     6001 tagcccgagc gcacctcgtc tgcctccggc aagcaccgaa ggtggtcgtc cgctgcagat
     6061 tgttcgtgaa accgttcgtc tgattgcaga acgtggtttt catgcagttc gtgttgcaga
     6121 tattgcagca gcatgtcata ccagcaccgc agcaattcat tatcattttc cgggtcgtga
     6181 tgaactgctg gaagcagcag ttcgttggtg tatggatgaa gatacccgtc gtcgtgcaga
     6241 tgcaaccgca ggcgcacgtc atgccggtga tgagctgcgt ctgctgattg aactgcagac
     6301 accgcgtacc gaacagcagc gtcgtcagtg gtgtgtttgg ctggatctgt gggcagaagc
     6361 agcacgtagc accaccgttg gtcagctgca tgttgaatat tatcgtcagt ggcgtggcac
     6421 cgttgcagat gttattcgtc gtggtgttgg tcagggtgtt tttcgtccgg ttgatgcaga
     6481 tggtgcagca ctgaccctga ccgcactgat tgatggtctg gcaagccagg ttctggcaac
     6541 cgctccgggt catccgggta caggtgcaca gaccatgcat gatgcactga ttgcacatgt
     6601 tagcgcatgt ctggcagcac cggcagcaga ttaactaggg cccatacccc caggcatcaa
     6661 ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg
     6721 aacgctctct actagagtca cactggctca ccttcgggtg ggcctttctg cgtttatacg
     6781 acgtacggtg gaacgagccg tcaattgtct gattcgttac caattgacac taactgacgg
     6841 ttcagttagg ttgctagcta ctagagaaag aggagaaata ctagatggtg agccgtcaat
     6901 tgtctgattc gttaccaatt gacactaact gctgttcagt taggttgcta gcaaagagga
     6961 gaaatactag atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     7021 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     7081 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     7141 ctggcccacc ctcgtgacca ccttcggcta cggcctgcaa tgcttcgccc gctaccccga
     7201 ccacatgaag ctgcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     7261 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     7321 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     7381 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     7441 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     7501 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     7561 cgacaaccac tacctgagct accagtccgc cctgagcaaa gaccccaacg agaagcgcga
     7621 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     7681 gtacaagtaa
//
