LOCUS       sequence-73297-e        7383 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 7383)
  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 7383)
  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-AmtR
FEATURES             Location/Qualifiers
     source          1..7383
                     /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      781..852
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      868..895
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             973..984
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(991..993,994..996,997..1710)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1036..1057)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1297..1316)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1644..1665
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1750..1767)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1920..2006
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2098..2125
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2144..2235
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2236..2304,2305..3096)
                     /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      3138..3593
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3225..3244)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4424..4441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4558..5103)
                     /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(4638..4657)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5175..5194
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(6169..7251)
                     /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     7213..7232
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc acaattctat
       61 ggactatgtt tgaaagagag aatactagat ggcaggcgca gttggtcgtc cgcgtcgtag
      121 tgcaccgcgt cgtgcaggta aaaatccgcg tgaagaaatt ctggatgcaa gcgcagaact
      181 gtttacccgt cagggttttg caaccaccag tacccatcag attgcagatg cagttggtat
      241 tcgtcaggca agcctgtatt atcattttcc gagcaaaacc gaaatctttc tgaccctgct
      301 gaaaagcacc gttgaaccga gcaccgttct ggcagaagat ctgagcaccc tggatgcagg
      361 tccggaaatg cgtctgtggg caattgttgc aagcgaagtt cgtctgctgc tgagcaccaa
      421 atggaatgtt ggtcgtctgt atcagctgcc gattgttggt agcgaagaat ttgcagaata
      481 tcatagccag cgtgaagcac tgaccaatgt ttttcgtgat ctggcaaccg aaattgttgg
      541 tgatgatccg cgtgcagaac tgccgtttca tattaccatg agcgttattg aaatgcgtcg
      601 caatgatggt aaaattccga gtccgctgag cgcagatagc ctgccggaaa ccgcaattat
      661 gctggcagat gcaagcctgg cagttctggg tgcaccgctg cctgcagatc gtgttgaaaa
      721 aaccctggaa ctgattaaac aggcagatgc aaaataacta gggcccatac ccccaggcat
      781 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
      841 gtgaacgctc tctactagag tcacactggc tcaccttcgg gtgggccttt ctgcgtttat
      901 acgacgtacg gtggaagatt cgttaccaat tgacagtttc tatcgatcta tagataatgc
      961 tagctactag agaaagagga gaaatactag atggtgagca agggcgagga gctgttcacc
     1021 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     1081 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     1141 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta cggcctgcaa
     1201 tgcttcgccc gctaccccga ccacatgaag ctgcacgact tcttcaagtc cgccatgccc
     1261 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     1321 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     1381 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     1441 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1501 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1561 gacggccccg tgctgctgcc cgacaaccac tacctgagct accagtccgc cctgagcaaa
     1621 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1681 actctcggca tggacgagct gtacaagtaa taagcttggc tgttttggcg gatgagagaa
     1741 gattttcagc ctgatacaga ttaaatcaga acgcagaagc ggtctgataa aacagaattt
     1801 gcctggcggc agtagcgcgg tggtcccacc tgaccccatg ccgaactcag aagtgaaacg
     1861 ccgtagcgcc gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc
     1921 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     1981 tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac
     2041 ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga
     2101 aggccatcct gacggatggc ctttttgcgt ttctacaaac tcttttgttt atttttctaa
     2161 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     2221 tgaaaaagga agactatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     2281 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2341 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2401 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2461 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     2521 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2581 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2641 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2701 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2761 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2821 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2881 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2941 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     3001 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     3061 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     3121 atactttaga ttgatttacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3181 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3241 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3301 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga
     3361 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3421 cacgttcttt aatagtggac tcttgttcca aacttgaaca acactcaacc ctatctcggg
     3481 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3541 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa tttaaaagga
     3601 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     3661 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     3721 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     3781 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     3841 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3901 cgcctacata cctcgctctg ctaatcctgt taccagtcag gcatttgaga agcacacggt
     3961 cacactgctt ccggtagtca ataaaccggt aaaccagcaa tagacataag cggctattta
     4021 acgaccctgc cctgaaccga cgaccgggtc gaatttgctt tcgaatttct gccattcatc
     4081 cgcttattat cacttattca ggcgtagcac caggcgttta agggcaccaa taactgcctt
     4141 aaaaaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca ttaagcattc
     4201 tgccgacatg gaagccatca cagacggcat gatgaacctg aatcgccagc ggcatcagca
     4261 ccttgtcgcc ttgcgtataa tatttgccca tggctagcgg agtgtatact ggcttactat
     4321 gttggcactg atgagggtgt cagtgaagtg cttcatgtgg caggagaaaa aaggctgcac
     4381 cggtgcgtca gcagaatatg tgatacagga tatattccgc ttcctcgctc actgactcgc
     4441 tacgctcggt cgttcgactg cggcgagcgg aaatggctta cgaacggggc ggagatttcc
     4501 tggaagatgc caggaagata cttaacaggg aagtgagagg gccgcggcaa agccgttttt
     4561 ccataggctc cgcccccctg acaagcatca cgaaatctga cgctcaaatc agtggtggcg
     4621 aaacccgaca ggactataaa gataccaggc gtttccccct ggcggctccc tcgtgcgctc
     4681 tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc gtttgtctca
     4741 ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac tgtatgcacg
     4801 aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     4861 cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt agaggagtta
     4921 gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg tgactgcgct
     4981 cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt cgaaaaaccg
     5041 ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc aaaacgatct
     5101 caagaagatc atcttattaa tcagataaaa tatttgctca tgagcccgaa gtggcgagcc
     5161 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     5221 gtgatgccgg ccacgatgcg tccggcgtag aggatctgct catgtttgac agcttatcat
     5281 cgatgcataa tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc tatgctactc
     5341 cctcgagccg tcaattgtct gattcgttac caattattaa tttttaaagt atgggcaatc
     5401 aattgctcct gttaaaattg ctttagaaat actttggcag cggtttgttg tattgagttt
     5461 catttgcgca ttggttaaat ggaaagtgac agtacgctca ctgcaaccta atatttttga
     5521 aatatcccaa gagctttttc cttcgcatgc ccacgctaaa cattcttttt ctcttttggt
     5581 taaatcgttg tttgatttat tatttgctat atttattttt cgataattat caactagaga
     5641 aggaacaatt aatggtatgt tcatacacgc atgtaaaaat aaactatcta tatagttgtc
     5701 tttttctgaa tgtgcaaaac taagcattcc gaagccattg ttagccgtat gaatagggaa
     5761 actaaaccca gtgataagac ctgatgtttt cgcttcttta attacatttg gagatttttt
     5821 atttacagca ttgttttcaa atatattcca attaattggt gaatgattgg agttagaata
     5881 atctactata ggatcatatt ttattaaatt agcgtcatca taatattgcc tccatttttt
     5941 agggtaatta tctaggattg aaatatcaga tttaaccata gaatgaggat aaatgatcgc
     6001 gagtaaataa tattcacaat gtaccatttt agtcatatca gataagcatt gattaatatc
     6061 attattgctt ctacaagctt taattttatt aattattctg tatgtgtcgt cggcatttat
     6121 gtttttcatc gaggcctttc ctgtgtgacc cgctggaata ggatcttatc actgcccgct
     6181 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     6241 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga ctggcaacag
     6301 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     6361 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctatc
     6421 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     6481 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     6541 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     6601 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     6661 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     6721 caatgcgacc agatgctcca cgcccagtcg cgtaccgtcc tcatgggaga aaataatact
     6781 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     6841 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     6901 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     6961 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     7021 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     7081 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     7141 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     7201 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca tattcaccac
     7261 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc
     7321 gatggcgcgc cgcttcgtca ggccacatag ctttcttgtt ctgatcggaa cgatcgttgg
     7381 ctg
//
