LOCUS       sequence-198050-        9437 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Bacterial expression plasmid for SpCas9∆REC3, REC2 FRET variant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9437)
  AUTHORS   Chen JS, Dagdas YS, Kleinstiver BP, Welch MM, Sousa AA, Harrington 
            LB, Sternberg SH, Joung JK, Yildiz A, Doudna JA
  TITLE     Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.
  JOURNAL   Nature. 2017 Sep 20. doi: 10.1038/nature24268.
  PUBMED    28931002
REFERENCE   2  (bases 1 to 9437)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2017 Sep 20. doi: 10.1038/nature24268."
COMMENT     Sequence Label: pJSC057 - Bacterial expression plasmid for
            SpC...
FEATURES             Location/Qualifiers
     source          1..9437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     139..158
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     242..261
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        242..260
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             349..371
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             391..420
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             430..1530
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLE
                     EKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKL
                     IAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIA
                     ADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGE
                     TAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL
                     ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF
                     WYAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1504..1527
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1585..1605
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5208..5226)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5222..5269
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5635..5640,5641..5657,5658..5663))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5690..5708)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5776..5880
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5881..5949,5950..6741)
                     /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(6099..6118)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6912..7500
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7401..7420
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7654..7671
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7686..7825
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7751..7770)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7911..7933
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(7927..8118)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
       61 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
      121 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
      181 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
      241 ttaatacgac tcactatagg gagaccacaa cggtttccct ctagtgccgg ctccggagag
      301 ctctttaatt aagcggccgc cctgcaggac tcgagttcta gaaataattt tgtttaactt
      361 taagaaggag atatacatat gaaatcttct caccatcacc atcaccatca ccatcaccat
      421 ggttcttcta tgaaaatcga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc
      481 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc
      541 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc
      601 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg
      661 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc
      721 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt
      781 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat
      841 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc
      901 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac
      961 gacattaaag acgtgggcgt ggataacgct ggcgcaaaag cgggtctgac cttcctggtt
     1021 gacctgatta aaaacaaaca catgaatgca gacaccgatt actccatcgc agaagctgcc
     1081 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac
     1141 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa
     1201 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca
     1261 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac
     1321 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt
     1381 attgccgcca ctatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg
     1441 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact
     1501 gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cgaacaacaa caacaataac
     1561 aataacaaca acctcgggat cgaggaaaac ctgtacttcc aatccaatgc aatggataag
     1621 aaatactcaa taggcttaga tatcggcaca aatagcgtcg gatgggcggt gatcactgat
     1681 gaatataagg ttccgtctaa aaagttcaag gttctgggaa atacagaccg ccacagtatc
     1741 aaaaaaaatc ttataggggc tcttttattt gacagtggag agacagcgtg tgcgactcgt
     1801 ctcaaacgga cagctcgtag aaggtataca cgtcggaaga atcgtatttc ttatctacag
     1861 gagatttttt caaatgagat ggcgaaagta gatgatagtt tctttcatcg acttgaagag
     1921 tcttttttgg tggaagaaga caagaagcat gaacgtcatc ctatttttgg aaatatagta
     1981 gatgaagttg cttatcatga gaaatatcca actatctatc atctgcgaaa aaaattggta
     2041 gattctactg ataaagcgga tttgcgctta atctatttgg ccttagcgca tatgattaag
     2101 tttcgtggtc attttttgat tgagggagat ttaaatcctg ataatagtga tgtggacaaa
     2161 ctatttatcc agttggtaca aacctacaat caattatttg aagaaaaccc tattaacgca
     2221 agtggagtag atgctaaagc gattctttct gcacgattga gtaaatcaag acgattagaa
     2281 aatctcattg ctcagctccc cggtgagaag aaaaatggct tatttgggaa tctcattgct
     2341 ttgtcattgg gtttgacccc taattttaaa tcaaattttg atttggcaga agatgctaaa
     2401 ttacagcttt caaaagatac ttacgattgt gatttagata atttattggc gcaaattgga
     2461 gatcaatatg ctgatttgtt tttggcagct aagaatttat cagatgctat tttactttca
     2521 gatatcctaa gagtaaatac tgaaataact aaggctcccc tatcagcttc aatgattaaa
     2581 cgctacgatg aacatcatca agacttgact cttttaaaag ctttagttcg acaacaactt
     2641 ccagaaaagt ataaagaaat cttttttgat caatcaaaaa acggatatgc aggttatatt
     2701 gatgggggag ctagccaaga agaattttat aaatttatca aaccaatttt agaaaaaatg
     2761 gatggtactg aggaattatt ggtgaaacta aatcgtgaag atttgctgcg caagcaacgg
     2821 acctttgaca acggctctat tccccatcaa attcacttgg gtgagctgca tgctattttg
     2881 agaagacaag aagactttta tccattttta aaagacaatc gtgagaagat tgaaaaaatc
     2941 ttgacttttc gaattcctta ttatgttggt ccattggcgc gtggcaatag tcgttttgca
     3001 tggatgactc ggaagtctga agaaacaatt accccatgga attttgaaga agttgtcgat
     3061 aaaggtgctt cagctcaatc atttattgaa cgcatgacaa acggtggaag tgtgtctgga
     3121 caaggcgata gtttacatga acatattgca aatttagctg gtagccctgc tattaaaaaa
     3181 ggtattttac agactgtaaa agttgttgat gaattggtca aagtaatggg gcggcataag
     3241 ccagaaaata tcgttattga aatggcacgt gaaaatcaga caactcaaaa gggccagaaa
     3301 aattcgcgag agcgtatgaa acgaatcgaa gaaggtatca aagaattagg aagtcagatt
     3361 cttaaagagc atcctgttga aaatactcaa ttgcaaaatg aaaagctcta tctctattat
     3421 ctccaaaatg gaagagacat gtatgtggac caagaattag atattaatcg tttaagtgat
     3481 tatgatgtcg atcacattgt tccacaaagt ttccttaaag acgattcaat agacaataag
     3541 gtcttaacgc gttctgataa aaatcgtggt aaatcggata acgttccaag tgaagaagta
     3601 gtcaaaaaga tgaaaaacta ttggagacaa cttctaaacg ccaagttaat cactcaacgt
     3661 aagtttgata atttaacgaa agctgaacgt ggaggtttga gtgaacttga taaagctggt
     3721 tttatcaaac gccaattggt tgaaactcgc caaatcacta agcatgtggc acaaattttg
     3781 gatagtcgca tgaatactaa atacgatgaa aatgataaac ttattcgaga ggttaaagtg
     3841 attaccttaa aatctaaatt agtttctgac ttccgaaaag atttccaatt ctataaagta
     3901 cgtgagatta acaattacca tcatgcccat gatgcgtatc taaatgccgt cgttggaact
     3961 gctttgatta agaaatatcc aaaacttgaa tcggagtttg tctatggtga ttataaagtt
     4021 tatgatgttc gtaaaatgat tgctaagtct gagcaagaaa taggcaaagc aaccgcaaaa
     4081 tatttctttt actctaatat catgaacttc ttcaaaacag aaattacact tgcaaatgga
     4141 gagattcgca aacgccctct aatcgaaact aatggggaaa ctggagaaat tgtctgggat
     4201 aaagggcgag attttgccac agtgcgcaaa gtattgtcca tgccccaagt caatattgtc
     4261 aagaaaacag aagtacagac aggcggattc tccaaggagt caattttacc aaaaagaaat
     4321 tcggacaagc ttattgctcg taaaaaagac tgggatccaa aaaaatatgg tggttttgat
     4381 agtccaacgg tagcttattc agtcctagtg gttgctaagg tggaaaaagg gaaatcgaag
     4441 aagttaaaat ccgttaaaga gttactaggg atcacaatta tggaaagaag ttcctttgaa
     4501 aaaaatccga ttgacttttt agaagctaaa ggatataagg aagttaaaaa agacttaatc
     4561 attaaactac ctaaatatag tctttttgag ttagaaaacg gtcgtaaacg gatgctggct
     4621 agtgccggag aattacaaaa aggaaatgag ctggctctgc caagcaaata tgtgaatttt
     4681 ttatatttag ctagtcatta tgaaaagttg aagggtagtc cagaagataa cgaacaaaaa
     4741 caattgtttg tggagcagca taagcattat ttagatgaga ttattgagca aatcagtgaa
     4801 ttttctaagc gtgttatttt agcagatgcc aatttagata aagttcttag tgcatataac
     4861 aaacatagag acaaaccaat acgtgaacaa gcagaaaata ttattcattt atttacgttg
     4921 acgaatcttg gagctcccgc tgcttttaaa tattttgata caacaattga tcgtaaacga
     4981 tatacgtcta caaaagaagt tttagatgcc actcttatcc atcaatccat cactggtctt
     5041 tatgaaacac gcattgattt gagtcagcta ggaggtgact aataacattg gaagtggata
     5101 acggatccgc gatcgcggcg cgccacctgg tggccggccg gtaccacgcg tgcgcgctga
     5161 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     5221 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     5281 aactatatcc ggatatccac aggacgggtg tggtcgccat gatcgcgtag tcgatagtgg
     5341 ctccaagtag cgaagcgagc aggactgggc ggcggccaaa gcggtcggac agtgctccga
     5401 gaacgggtgc gcatagaaat tgcatcaacg catatagcgc tagcagcacg ccatagtgac
     5461 tggcgatgct gtcggaatgg acgatatccc gcaagaggcc cggcagtacc ggcataacca
     5521 agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag
     5581 atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa
     5641 gcttatcgat gataagctgt caaacatgag aattcttgaa gacgaaaggg cctcgtgata
     5701 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     5761 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5821 tatccgctca tgagacaata accctgataa atgcttcaat aacattgaaa aaggaagagt
     5881 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5941 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     6001 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     6061 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     6121 cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     6181 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     6241 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     6301 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6361 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6421 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6481 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6541 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6601 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6661 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6721 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6781 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6841 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6901 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6961 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     7021 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     7081 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     7141 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     7201 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     7261 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     7321 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     7381 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     7441 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     7501 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7561 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     7621 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     7681 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     7741 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     7801 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     7861 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     7921 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     7981 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     8041 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     8101 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     8161 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     8221 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     8281 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     8341 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     8401 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     8461 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     8521 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     8581 ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg cggctgctgg agatggcgga
     8641 cgcgatggat atgttctgcc aagggttggt ttgcgcattc acagttctcc gcaagaattg
     8701 attggctcca attcttggag tggtgaatcc gttagcgagg tgccgccggc ttccattcag
     8761 gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac aaggtatagg
     8821 gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc ataaatcgcc
     8881 gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag cgatccttga
     8941 agctgtccct gatggtcgtc atctacctgc ctggacagca tggcctgcaa cgcgggcatc
     9001 ccgatgccgc cggaagcgag aagaatcata atggggaagg ccatccagcc tcgcgtcgcg
     9061 aacgccagca agacgtagcc cagcgcgtcg gccgccatgc cggcgataat ggcctgcttc
     9121 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     9181 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     9241 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     9301 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     9361 gctctcaagg gcatcggtcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     9421 agtagtaggt tgaggcc
//
