LOCUS       Exported                7804 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Allows inducible expression of gRNAs and Cas9 for plasmid curing..
ACCESSION   .
VERSION     .
KEYWORDS    pFREE_Amp
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7804)
  AUTHORS   Lauritsen I, Porse A, Sommer MOA, Norholm MHH
  TITLE     A versatile one-step CRISPR-Cas9 based approach to plasmid-curing.
  JOURNAL   Microb Cell Fact. 2017 Aug 2;16(1):135. doi: 
            10.1186/s12934-017-0748-z.
  PUBMED    28764701
REFERENCE   2  (bases 1 to 7804)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7804
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    1..19
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    26..44
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             130..4236
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     complement(4271..4289)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4285..4332
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             4466..5089
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(4539..4558)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     terminator      5207..5301
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(5330..6190)
                     /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     5953..5972
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6191..6254)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6298..6933)
                     /direction=LEFT
                     /label=ColA ori
                     /note="Plasmids containing the ColA origin of replication 
                     can be propagated in E. coli cells that contain additional 
                     plasmids with compatible origins."
     primer_bind     complement(6443..6462)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     misc_RNA        complement(7182..7260)
                     /label=tracrRNA
                     /note="trans-activating CRISPR RNA for the Streptococcus 
                     pyogenes CRISPR/Cas9 system"
     repeat_region   7432..7467
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   7498..7533
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   7564..7599
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   7630..7665
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
     repeat_region   7696..7731
                     /label=DR
                     /note="direct repeat for the Streptococcus pyogenes 
                     CRISPR/Cas system"
ORIGIN
        1 tccctatcag tgatagagat tgacatccct atcagtgata gagatactga gcacagaagg
       61 agatatacat ggataagaaa tactgagcac tatcagtgat agagatactg agcacagaag
      121 gagatataca tggataagaa atactcaata ggcttagata tcggcacaaa tagcgtcgga
      181 tgggcggtga tcactgatga atataaggtt ccgtctaaaa agttcaaggt tctgggaaat
      241 acagaccgcc acagtatcaa aaaaaatctt ataggggctc ttttatttga cagtggagag
      301 acagcggaag cgactcgtct caaacggaca gctcgtagaa ggtatacacg tcggaagaat
      361 cgtatttgtt atctacagga gattttttca aatgagatgg cgaaagtaga tgatagtttc
      421 tttcatcgac ttgaagagtc ttttttggtg gaagaagaca agaagcatga acgtcatcct
      481 atttttggaa atatagtaga tgaagttgct tatcatgaga aatatccaac tatctatcat
      541 ctgcgaaaaa aattggtaga ttctactgat aaagcggatt tgcgcttaat ctatttggcc
      601 ttagcgcata tgattaagtt tcgtggtcat tttttgattg agggagattt aaatcctgat
      661 aatagtgatg tggacaaact atttatccag ttggtacaaa cctacaatca attatttgaa
      721 gaaaacccta ttaacgcaag tggagtagat gctaaagcga ttctttctgc acgattgagt
      781 aaatcaagac gattagaaaa tctcattgct cagctccccg gtgagaagaa aaatggctta
      841 tttgggaatc tcattgcttt gtcattgggt ttgaccccta attttaaatc aaattttgat
      901 ttggcagaag atgctaaatt acagctttca aaagatactt acgatgatga tttagataat
      961 ttattggcgc aaattggaga tcaatatgct gatttgtttt tggcagctaa gaatttatca
     1021 gatgctattt tactttcaga tatcctaaga gtaaatactg aaataactaa ggctccccta
     1081 tcagcttcaa tgattaaacg ctacgatgaa catcatcaag acttgactct tttaaaagct
     1141 ttagttcgac aacaacttcc agaaaagtat aaagaaatct tttttgatca atcaaaaaac
     1201 ggatatgcag gttatattga tgggggagct agccaagaag aattttataa atttatcaaa
     1261 ccaattttag aaaaaatgga tggtactgag gaattattgg tgaaactaaa tcgtgaagat
     1321 ttgctgcgca agcaacggac ctttgacaac ggctctattc cccatcaaat tcacttgggt
     1381 gagctgcatg ctattttgag aagacaagaa gacttttatc catttttaaa agacaatcgt
     1441 gagaagattg aaaaaatctt gacttttcga attccttatt atgttggtcc attggcgcgt
     1501 ggcaatagtc gttttgcatg gatgactcgg aagtctgaag aaacaattac cccatggaat
     1561 tttgaagaag ttgtcgataa aggtgcttca gctcaatcat ttattgaacg catgacaaac
     1621 tttgataaaa atcttccaaa tgaaaaagta ctaccaaaac atagtttgct ttatgagtat
     1681 tttacggttt ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat gcgaaaacca
     1741 gcatttcttt caggtgaaca gaagaaagcc attgttgatt tactcttcaa aacaaatcga
     1801 aaagtaaccg ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg ttttgatagt
     1861 gttgaaattt caggagttga agatagattt aatgcttcat taggtaccta ccatgatttg
     1921 ctaaaaatta ttaaagataa agattttttg gataatgaag aaaatgaaga tatcttagag
     1981 gatattgttt taacattgac cttatttgaa gatagggaga tgattgagga aagacttaaa
     2041 acatatgctc acctctttga tgataaggtg atgaaacagc ttaaacgtcg ccgttatact
     2101 ggttggggac gtttgtctcg aaaattgatt aatggtatta gggataagca atctggcaaa
     2161 acaatattag attttttgaa atcagatggt tttgccaatc gcaattttat gcagctgatc
     2221 catgatgata gtttgacatt taaagaagac attcaaaaag cacaagtgtc tggacaaggc
     2281 gatagtttac atgaacatat tgcaaattta gctggtagcc ctgctattaa aaaaggtatt
     2341 ttacagactg taaaagttgt tgatgaattg gtcaaagtaa tggggcggca taagccagaa
     2401 aatatcgtta ttgaaatggc acgtgaaaat cagacaactc aaaagggcca gaaaaattcg
     2461 cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat taggaagtca gattcttaaa
     2521 gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta ttatctccaa
     2581 aatggaagag acatgtatgt ggaccaagaa ttagatatta atcgtttaag tgattatgat
     2641 gtcgatcaca ttgttccaca aagtttcctt aaagacgatt caatagacaa taaggtctta
     2701 acgcgttctg ataaaaatcg tggtaaatcg gataacgttc caagtgaaga agtagtcaaa
     2761 aagatgaaaa actattggag acaacttcta aacgccaagt taatcactca acgtaagttt
     2821 gataatttaa cgaaagctga acgtggaggt ttgagtgaac ttgataaagc tggttttatc
     2881 aaacgccaat tggttgaaac tcgccaaatc actaagcatg tggcacaaat tttggatagt
     2941 cgcatgaata ctaaatacga tgaaaatgat aaacttattc gagaggttaa agtgattacc
     3001 ttaaaatcta aattagtttc tgacttccga aaagatttcc aattctataa agtacgtgag
     3061 attaacaatt accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg aactgctttg
     3121 attaagaaat atccaaaact tgaatcggag tttgtctatg gtgattataa agtttatgat
     3181 gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc aaaatatttc
     3241 ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa tggagagatt
     3301 cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg ggataaaggg
     3361 cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat tgtcaagaaa
     3421 acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag aaattcggac
     3481 aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt tgatagtcca
     3541 acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc gaagaagtta
     3601 aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt tgaaaaaaat
     3661 ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt aatcattaaa
     3721 ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct ggctagtgcc
     3781 ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa ttttttatat
     3841 ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca aaaacaattg
     3901 tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag tgaattttct
     3961 aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata taacaaacat
     4021 agagacaaac caatacgtga acaagcagaa aatattattc atttatttac gttgacgaat
     4081 cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa acgatatacg
     4141 tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg tctttatgaa
     4201 acacgcattg atttgagtca gctaggaggt gactgagatc ccatgttacc ggtatcctag
     4261 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     4321 aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact gcttccggta
     4381 gtcaataaac cggtattgac gccaacgggt cacacgggta cagtgctagc agaaacatat
     4441 ccatgaaatc ccgccccgaa ttcatatgtc tagattagat aaaagtaaag tgattaacag
     4501 cgcattagag ctgcttaatg aggtcggaat cgaaggttta acaacccgta aactcgccca
     4561 gaagctaggt gtagagcagc ctacattgta ttggcatgta aaaaataagc gggctttgct
     4621 cgacgcctta gccattgaga tgttagatag gcaccatact cacttttgcc ctttagaagg
     4681 ggaaagctgg caagattttt tacgtaataa cgctaaaagt tttagatgtg ctttactaag
     4741 tcatcgcgat ggagcaaaag tacatttagg tacacggcct acagaaaaac agtatgaaac
     4801 tctcgaaaat caattagcct ttttatgcca acaaggtttt tcactagaga atgcattata
     4861 tgcactcagc gctgtggggc attttacttt aggttgcgta ttggaagatc aagagcatca
     4921 agtcgctaaa gaagaaaggg aaacacctac tactgatagt atgccgccat tattacgaca
     4981 agctatcgaa ttatttgatc accaaggtgc agagccagcc ttcttattcg gccttgaatt
     5041 gatcatatgc ggattagaaa aacaacttaa atgtgaaagt gggtcttaag caatagacat
     5101 aagcggctat ttaacgaccc tgccctgaac cgacgacaag ctgacgaccg ggtctccgca
     5161 agtggcactt ttcggggaaa tgtgcgcgga acccctatgt gcttggattc tcaccaataa
     5221 aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct gaggtcatta
     5281 ctggatctat caacaggagt ccaagcgagc tctaaacttg gtctgacagt taccaatgct
     5341 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5401 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5461 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5521 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5581 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5641 ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5701 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5761 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5821 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5881 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5941 cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     6001 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     6061 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6121 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6181 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6241 tgagcggata catatttgga cgtctaagaa accatacaaa taggcatgct agcgcagaaa
     6301 cgtcctagaa gatgccagga ggatacttag cagagagaca ataaggccgg agcgaagccg
     6361 tttttccata ggctccgccc ccctgacgaa catcacgaaa tctgacgctc aaatcagtgg
     6421 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctgatgg ctccctcttg
     6481 cgctctcctg ttcccgtcct gcggcgtccg tgttgtggtg gaggctttac ccaaatcacc
     6541 acgtcccgtt ccgtgtagac agttcgctcc aagctgggct gtgtgcaaga accccccgtt
     6601 cagcccgact gctgcgcctt atccggtaac tatcatcttg agtccaaccc ggaaagacac
     6661 gacaaaacgc cactggcagc agccattggt aactgagaat tagtggattt agatatcgag
     6721 agtcttgaag tggtggccta acagaggcta cactgaaagg acagtatttg gtatctgcgc
     6781 tccactaaag ccagttacca ggttaagcag ttccccaact gacttaacct tcgatcaaac
     6841 cgcctcccca ggcggttttt tcgtttacag agcaggagat tacgacgatc gtaaaaggat
     6901 ctcaagaaga tcctttacgg attcccgaca ccatcactct agatttcagt gcaatttatc
     6961 tcttcaaatg tagcacctga agtcagcccc atacgatata agttgtaatt ctcatgttag
     7021 tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc
     7081 gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc acaccgacta
     7141 gcgaaaaaac cccgccgaag cggggttttt tgcgaaaaaa agcaccgact cggtgccact
     7201 ttttcaagtt gataacggac tagccttatt ttaacttgct atgctgtttt gaatggttcc
     7261 gctagcactg tacctaggac tgagctagcc gtcaaaagca gcgtatatac catggccaca
     7321 attcagcaaa ttgtgaacat catcacgttc atctttccct ggttgccaat ggcccatttt
     7381 cctgtcagta acgagaaggt cgcgaattca ggcgcttttt agactggtcg tgttttagag
     7441 ctatgctgtt ttgaatggtc ccaaaacatg aactagcgat tagtcgctat gacttaagtt
     7501 ttagagctat gctgttttga atggtcccaa aacaaccaca ctagagaaca tactggctaa
     7561 atagttttag agctatgctg ttttgaatgg tcccaaaacg gttggactca agacgatagt
     7621 taccggatag ttttagagct atgctgtttt gaatggtccc aaaacggcga aacccgacag
     7681 gactataaag ataccgtttt agagctatgc tgttttgaat ggtcccaaaa caaaaaaaaa
     7741 ccccgcccct gacagggcgg ggtttttttt ttaattaaag tctggtcctc gagtctggtt
     7801 ataa
//
