LOCUS       Exported                7289 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pRF-BM3R1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7289)
  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 7289)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7289
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        3..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      684..755
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      771..798
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     RBS             879..890
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             897..1616
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(942..963)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1203..1222)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1550..1571
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1656..1673)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      1826..1912
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2004..2031
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     promoter        2050..2141
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2142..3002
                     /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"
     primer_bind     complement(2360..2379)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3044..3499
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3131..3150)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(4330..4347)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4464..5009)
                     /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(4544..4563)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5081..5100
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(6075..7157)
                     /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     7119..7138
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc acaattctat
       61 ggactatgtt ttaactacta gatggaaagc accccgacca aacagaaagc aatttttagc
      121 gcaagcctgc tgctgtttgc agaacgtggt tttgatgcaa ccaccatgcc gatgattgca
      181 gaaaatgcaa aagttggtgc aggcaccatt tatcgctatt tcaaaaacaa agaaagcctg
      241 gtgaacgaac tgtttcagca gcatgttaat gaatttctgc agtgtattga aagcggtctg
      301 gcaaatgaac gtgatggtta tcgtgatggc tttcatcaca tttttgaagg tatggtgacc
      361 tttaccaaaa atcatccgcg tgcactgggt tttatcaaaa cccatagcca gggcaccttt
      421 ctgaccgaag aaagccgtct ggcatatcag aaactggttg aatttgtgtg cacctttttt
      481 cgtgaaggtc agaaacaggg tgtgattcgt aatctgccgg aaaatgcact gattgcaatt
      541 ctgtttggca gctttatgga agtgtatgaa atgatcgaga acgattatct gagcctgacc
      601 gatgaactgc tgaccggtgt tgaagaaagc ctgtgggcag cactgagccg tcagagctaa
      661 ctagggccca tacccccagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
      721 ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact ggctcacctt
      781 cgggtgggcc tttctgcgtt tatacgacgt acggtggaat ctgattcgtt accaattgac
      841 ggaatgaacg ttcattccga taatgctagc tactagagaa agaggagaaa tactagatgg
      901 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
      961 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     1021 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     1081 tgaccacctt cggctacggc ctgcaatgct tcgcccgcta ccccgaccac atgaagctgc
     1141 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     1201 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     1261 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     1321 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     1381 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     1441 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     1501 tgagctacca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     1561 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaataag
     1621 cttggctgtt ttggcggatg agagaagatt ttcagcctga tacagattaa atcagaacgc
     1681 agaagcggtc tgataaaaca gaatttgcct ggcggcagta gcgcggtggt cccacctgac
     1741 cccatgccga actcagaagt gaaacgccgt agcgccgatg gtagtgtggg gtctccccat
     1801 gcgagagtag ggaactgcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc
     1861 ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg agtaggacaa atccgccggg
     1921 agcggatttg aacgttgcga agcaacggcc cggagggtgg cgggcaggac gcccgccata
     1981 aactgccagg catcaaatta agcagaaggc catcctgacg gatggccttt ttgcgtttct
     2041 acaaactctt ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     2101 aaccctgata aatgcttcaa taatattgaa aaaggaagac tatgagtatt caacatttcc
     2161 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     2221 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     2281 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     2341 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac gccgggcaag
     2401 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     2461 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     2521 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     2581 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     2641 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca atggcaacaa
     2701 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     2761 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     2821 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     2881 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     2941 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     3001 aactgtcaga ccaagtttac tcatatatac tttagattga tttacgcgcc ctgtagcggc
     3061 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     3121 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     3181 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3241 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3301 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3361 tgaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     3421 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3481 atattaacgt ttacaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     3541 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     3601 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     3661 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     3721 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg
     3781 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     3841 agtcaggcat ttgagaagca cacggtcaca ctgcttccgg tagtcaataa accggtaaac
     3901 cagcaataga cataagcggc tatttaacga ccctgccctg aaccgacgac cgggtcgaat
     3961 ttgctttcga atttctgcca ttcatccgct tattatcact tattcaggcg tagcaccagg
     4021 cgtttaaggg caccaataac tgccttaaaa aaattacgcc ccgccctgcc actcatcgca
     4081 gtactgttgt aattcattaa gcattctgcc gacatggaag ccatcacaga cggcatgatg
     4141 aacctgaatc gccagcggca tcagcacctt gtcgccttgc gtataatatt tgcccatggc
     4201 tagcggagtg tatactggct tactatgttg gcactgatga gggtgtcagt gaagtgcttc
     4261 atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat acaggatata
     4321 ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat
     4381 ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta acagggaagt
     4441 gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa
     4501 atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4561 ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt
     4621 ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt
     4681 tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc
     4741 cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc
     4801 cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg
     4861 aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg
     4921 tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag
     4981 agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt
     5041 tgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     5101 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     5161 tctgctcatg tttgacagct tatcatcgat gcataatgtg cctgtcaaat ggacgaagca
     5221 gggattctgc aaaccctatg ctactccctc gagccgtcaa ttgtctgatt cgttaccaat
     5281 tattaatttt taaagtatgg gcaatcaatt gctcctgtta aaattgcttt agaaatactt
     5341 tggcagcggt ttgttgtatt gagtttcatt tgcgcattgg ttaaatggaa agtgacagta
     5401 cgctcactgc aacctaatat ttttgaaata tcccaagagc tttttccttc gcatgcccac
     5461 gctaaacatt ctttttctct tttggttaaa tcgttgtttg atttattatt tgctatattt
     5521 atttttcgat aattatcaac tagagaagga acaattaatg gtatgttcat acacgcatgt
     5581 aaaaataaac tatctatata gttgtctttt tctgaatgtg caaaactaag cattccgaag
     5641 ccattgttag ccgtatgaat agggaaacta aacccagtga taagacctga tgttttcgct
     5701 tctttaatta catttggaga ttttttattt acagcattgt tttcaaatat attccaatta
     5761 attggtgaat gattggagtt agaataatct actataggat catattttat taaattagcg
     5821 tcatcataat attgcctcca ttttttaggg taattatcta ggattgaaat atcagattta
     5881 accatagaat gaggataaat gatcgcgagt aaataatatt cacaatgtac cattttagtc
     5941 atatcagata agcattgatt aatatcatta ttgcttctac aagctttaat tttattaatt
     6001 attctgtatg tgtcgtcggc atttatgttt ttcatcgagg cctttcctgt gtgacccgct
     6061 ggaataggat cttatcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     6121 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     6181 cttttcacca gtgagactgg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     6241 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     6301 aacggcggga tataacatga gctatcttcg gtatcgtcgt atcccactac cgagatatcc
     6361 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     6421 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     6481 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     6541 gtgagatatt tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc
     6601 gctaacagcg cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta
     6661 ccgtcctcat gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat
     6721 aacgccggaa cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga
     6781 tagttaatga tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag
     6841 gcttcgacgc cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg
     6901 cgagatttaa tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca
     6961 acgccaatca gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa
     7021 ttcagctccg ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc
     7081 tggttcacca cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat
     7141 aacgttactg gtttcatatt caccaccctg aattgactct cttccgggcg ctatcatgcc
     7201 ataccgcgaa aggttttgcg ccattcgatg gcgcgccgct tcgtcaggcc acatagcttt
     7261 cttgttctga tcggaacgat cgttggctg
//
