LOCUS       Exported                6864 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  This construct encodes for two units. The first contains a 
            constitutively expressed dCas9 protein. The second contains an sgRNA
            cassette under the control of the burden-responsive htpG1 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    Biomolecular FeedBack (FB)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6864)
  AUTHORS   Ceroni F, Boo A, Furini S, Gorochowski TE, Borkowski O, Ladak YN, 
            Awan AR, Gilbert C, Stan GB, Ellis T
  TITLE     Burden-driven feedback control of gene expression.
  JOURNAL   Nat Methods. 2018 Mar 26. pii: nmeth.4635. doi: 10.1038/nmeth.4635.
  PUBMED    29578536
REFERENCE   2  (bases 1 to 6864)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6864
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      3136..3207
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3223..3250
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_RNA        3366..3441
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     terminator      3522..3608
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      complement(3777..4322)
                     /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(3857..3876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      4436..4530
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(4560..5420)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5183..5202
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5421..5525)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5593..5611
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     terminator      5644..5701
                     /label=his operon terminator
                     /note="This putative transcriptin terminator from the E. 
                     coli his operon has a 2-bp deletion introduced during 
                     synthesis. Its efficiency has not been determined."
     CDS             5870..3112
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
ORIGIN
        1 gactctttta aaagctttag ttcgacaaca acttccagaa aagtataaag aaatcttttt
       61 tgatcaatca aaaaacggat atgcaggtta tattgatggg ggagctagcc aagaagaatt
      121 ttataaattt atcaaaccaa ttttagaaaa aatggatggt actgaggaat tattggtgaa
      181 actaaatcgt gaagatttgc tgcgcaagca acggaccttt gacaacggct ctattcccca
      241 tcaaattcac ttgggtgagc tgcatgctat tttgagaaga caagaagact tttatccatt
      301 tttaaaagac aatcgtgaga agattgaaaa aatcttgact tttcgaattc cttattatgt
      361 tggtccattg gcgcgtggca atagtcgttt tgcatggatg actcggaagt ctgaagaaac
      421 aattacccca tggaattttg aagaagttgt cgataaaggt gcttcagctc aatcatttat
      481 tgaacgcatg acaaactttg ataaaaatct tccaaatgaa aaagtactac caaaacatag
      541 tttgctttat gagtatttta cggtttataa cgaattgaca aaggtcaaat atgttactga
      601 aggaatgcga aaaccagcat ttctttcagg tgaacagaag aaagccattg ttgatttact
      661 cttcaaaaca aatcgaaaag taaccgttaa gcaattaaaa gaagattatt tcaaaaaaat
      721 agaatgtttt gatagtgttg aaatttcagg agttgaagat agatttaatg cttcattagg
      781 tacctaccat gatttgctaa aaattattaa agataaagat tttttggata atgaagaaaa
      841 tgaagatatc ttagaggata ttgttttaac attgacctta tttgaagata gggagatgat
      901 tgaggaaaga cttaaaacat atgctcacct ctttgatgat aaggtgatga aacagcttaa
      961 acgtcgccgt tatactggtt ggggacgttt gtctcgaaaa ttgattaatg gtattaggga
     1021 taagcaatct ggcaaaacaa tattagattt tttgaaatca gatggttttg ccaatcgcaa
     1081 ttttatgcag ctgatccatg atgatagttt gacatttaaa gaagacattc aaaaagcaca
     1141 agtgtctgga caaggcgata gtttacatga acatattgca aatttagctg gtagccctgc
     1201 tattaaaaaa ggtattttac agactgtaaa agttgttgat gaattggtca aagtaatggg
     1261 gcggcataag ccagaaaata tcgttattga aatggcacgt gaaaatcaga caactcaaaa
     1321 gggccagaaa aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca aagaattagg
     1381 aagtcagatt cttaaagagc atcctgttga aaatactcaa ttgcaaaatg aaaagctcta
     1441 tctctattat ctccaaaatg gaagagacat gtatgtggac caagaattag atattaatcg
     1501 tttaagtgat tatgatgtcg atgccattgt tccacaaagt ttccttaaag acgattcaat
     1561 agacaataag gtcttaacgc gttctgataa aaatcgtggt aaatcggata acgttccaag
     1621 tgaagaagta gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg ccaagttaat
     1681 cactcaacgt aagtttgata atttaacgaa agctgaacgt ggaggtttga gtgaacttga
     1741 taaagctggt tttatcaaac gccaattggt tgaaactcgc caaatcacta agcatgtggc
     1801 acaaattttg gatagtcgca tgaatactaa atacgatgaa aatgataaac ttattcgaga
     1861 ggttaaagtg attaccttaa aatctaaatt agtttctgac ttccgaaaag atttccaatt
     1921 ctataaagta cgtgagatta acaattacca tcatgcccat gatgcgtatc taaatgccgt
     1981 cgttggaact gctttgatta agaaatatcc aaaacttgaa tcggagtttg tctatggtga
     2041 ttataaagtt tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa taggcaaagc
     2101 aaccgcaaaa tatttctttt actctaatat catgaacttc ttcaaaacag aaattacact
     2161 tgcaaatgga gagattcgca aacgccctct aatcgaaact aatggggaaa ctggagaaat
     2221 tgtctgggat aaagggcgag attttgccac agtgcgcaaa gtattgtcca tgccccaagt
     2281 caatattgtc aagaaaacag aagtacagac aggcggattc tccaaggagt caattttacc
     2341 aaaaagaaat tcggacaagc ttattgctcg taaaaaagac tgggatccaa aaaaatatgg
     2401 tggttttgat agtccaacgg tagcttattc agtcctagtg gttgctaagg tggaaaaagg
     2461 gaaatcgaag aagttaaaat ccgttaaaga gttactaggg atcacaatta tggaaagaag
     2521 ttcctttgaa aaaaatccga ttgacttttt agaagctaaa ggatataagg aagttaaaaa
     2581 agacttaatc attaaactac ctaaatatag tctttttgag ttagaaaacg gtcgtaaacg
     2641 gatgctggct agtgccggag aattacaaaa aggaaatgag ctggctctgc caagcaaata
     2701 tgtgaatttt ttatatttag ctagtcatta tgaaaagttg aagggtagtc cagaagataa
     2761 cgaacaaaaa caattgtttg tggagcagca taagcattat ttagatgaga ttattgagca
     2821 aatcagtgaa ttttctaagc gtgttatttt agcagatgcc aatttagata aagttcttag
     2881 tgcatataac aaacatagag acaaaccaat acgtgaacaa gcagaaaata ttattcattt
     2941 atttacgttg acgaatcttg gagctcccgc tgcttttaaa tattttgata caacaattga
     3001 tcgtaaacga tatacgtcta caaaagaagt tttagatgcc actcttatcc atcaatccat
     3061 cactggtctt tatgaaacac gcattgattt gagtcagcta ggaggtgact aactcgagta
     3121 aggatctcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     3181 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     3241 cctttctgcg tttataattt aaatggccgc ctaggccacg agagcaggat cacctgctct
     3301 cgcttgaaat tattctccct tgtccccatc tctcccatta attaagagtt gcgataaaaa
     3361 gcgtcgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     3421 aaagtggcac cgagtcggtg ctttttttat ggcgcgccta cagcggccgc gattatcaaa
     3481 aaggatcttc ggggatccca tggtacgcgt gctagaggca tcaaataaaa cgaaaggctc
     3541 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta
     3601 ggacaaatcc gccgccctag acctagggat atattccgct tcctcgctca ctgactcgct
     3661 acgctcggtc gttcgactgc ggcgagcgga aatggcttac gaacggggcg gagatttcct
     3721 ggaagatgcc aggaagatac ttaacaggga agtgagaggg ccgcggcaaa gccgtttttc
     3781 cataggctcc gcccccctga caagcatcac gaaatctgac gctcaaatca gtggtggcga
     3841 aacccgacag gactataaag ataccaggcg tttccccctg gcggctccct cgtgcgctct
     3901 cctgttcctg cctttcggtt taccggtgtc attccgctgt tatggccgcg tttgtctcat
     3961 tccacgcctg acactcagtt ccgggtaggc agttcgctcc aagctggact gtatgcacga
     4021 accccccgtt cagtccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4081 ggaaagacat gcaaaagcac cactggcagc agccactggt aattgattta gaggagttag
     4141 tcttgaagtc atgcgccggt taaggctaaa ctgaaaggac aagttttggt gactgcgctc
     4201 ctccaagcca gttacctcgg ttcaaagagt tggtagctca gagaaccttc gaaaaaccgc
     4261 cctgcaaggc ggttttttcg ttttcagagc aagagattac gcgcagacca aaacgatctc
     4321 aagaagatca tcttattaat cagataaaat atttctagat ttcagtgcaa tttatctctt
     4381 caaatgtagc acctgaagtc agccccatac gatataagtt gttactagtg cttggattct
     4441 caccaataaa aaacgcccgg cggcaaccga gcgttctgaa caaatccaga tggagttctg
     4501 aggtcattac tggatctatc aacaggagtc caagcgagct cgtaaacttg gtctgacagt
     4561 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4621 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4681 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     4741 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     4801 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     4861 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     4921 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4981 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5041 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5101 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5161 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5221 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5281 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5341 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5401 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5461 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5521 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     5581 ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcacctcga ggcggccgcc
     5641 ccttccggca aaaaagggca aggtgtcacc accctgccct ttttctttaa aaccgaaaag
     5701 attacttcgc gttatgcagg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5761 gcggcgagcg gtatcagctc actcaaaggt cacctcgagt ttacggctag ctcagtccta
     5821 ggtattgtgc tagcaagaat tcaaaagctc tacagaggag aaaggatata tggataagaa
     5881 atactcaata ggcttagcta tcggcacaaa tagcgtcgga tgggcggtga tcactgatga
     5941 atataaggtt ccgtctaaaa agttcaaggt tctgggaaat acagaccgcc acagtatcaa
     6001 aaaaaatctt ataggggctc ttttatttga cagtggagag acagcggaag cgactcgtct
     6061 caaacggaca gctcgtagaa ggtatacacg tcggaagaat cgtatttgtt atctacagga
     6121 gattttttca aatgagatgg cgaaagtaga tgatagtttc tttcatcgac ttgaagagtc
     6181 ttttttggtg gaagaagaca agaagcatga acgtcatcct atttttggaa atatagtaga
     6241 tgaagttgct tatcatgaga aatatccaac tatctatcat ctgcgaaaaa aattggtaga
     6301 ttctactgat aaagcggatt tgcgcttaat ctatttggcc ttagcgcata tgattaagtt
     6361 tcgtggtcat tttttgattg agggagattt aaatcctgat aatagtgatg tggacaaact
     6421 atttatccag ttggtacaaa cctacaatca attatttgaa gaaaacccta ttaacgcaag
     6481 tggagtagat gctaaagcga ttctttctgc acgattgagt aaatcaagac gattagaaaa
     6541 tctcattgct cagctccccg gtgagaagaa aaatggctta tttgggaatc tcattgcttt
     6601 gtcattgggt ttgaccccta attttaaatc aaattttgat ttggcagaag atgctaaatt
     6661 acagctttca aaagatactt acgatgatga tttagataat ttattggcgc aaattggaga
     6721 tcaatatgct gatttgtttt tggcagctaa gaatttatca gatgctattt tactttcaga
     6781 tatcctaaga gtaaatactg aaataactaa ggctccccta tcagcttcaa tgattaaacg
     6841 ctacgatgaa catcatcaag actt
//
