LOCUS       sequence-197227-        9437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for SpCas9∆REC3 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 Dec 15, 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: pSHS273 - Bacterial expression plasmid for
            SpC...
FEATURES             Location/Qualifiers
     source          1..9437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     376..395
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     479..498
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        479..497
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             586..608
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             628..657
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             667..1767
                     /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     1741..1764
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1822..1842
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5445..5463)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5459..5506
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5872..5877,5878..5894,5895..5900))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(5927..5945)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6013..6117
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6118..6186,6187..6978)
                     /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(6336..6355)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7149..7737
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7638..7657
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7891..7908
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    7923..8062
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(7988..8007)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8148..8170
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8164..8355)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc
       61 cggcacctgt cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt
      121 catgccccgc gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg
      181 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
      241 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
      301 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
      361 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
      421 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
      481 atacgactca ctatagggag accacaacgg tttccctcta gtgccggctc cggagagctc
      541 tttaattaag cggccgccct gcaggactcg agttctagaa ataattttgt ttaactttaa
      601 gaaggagata tacatatgaa atcttctcac catcaccatc accatcacca tcaccatggt
      661 tcttctatga aaatcgaaga aggtaaactg gtaatctgga ttaacggcga taaaggctat
      721 aacggtctcg ctgaagtcgg taagaaattc gagaaagata ccggaattaa agtcaccgtt
      781 gagcatccgg ataaactgga agagaaattc ccacaggttg cggcaactgg cgatggccct
      841 gacattatct tctgggcaca cgaccgcttt ggtggctacg ctcaatctgg cctgttggct
      901 gaaatcaccc cggacaaagc gttccaggac aagctgtatc cgtttacctg ggatgccgta
      961 cgttacaacg gcaagctgat tgcttacccg atcgctgttg aagcgttatc gctgatttat
     1021 aacaaagatc tgctgccgaa cccgccaaaa acctgggaag agatcccggc gctggataaa
     1081 gaactgaaag cgaaaggtaa gagcgcgctg atgttcaacc tgcaagaacc gtacttcacc
     1141 tggccgctga ttgctgctga cgggggttat gcgttcaagt atgaaaacgg caagtacgac
     1201 attaaagacg tgggcgtgga taacgctggc gcgaaagcgg gtctgacctt cctggttgac
     1261 ctgattaaaa acaaacacat gaatgcagac accgattact ccatcgcaga agctgccttt
     1321 aataaaggcg aaacagcgat gaccatcaac ggcccgtggg catggtccaa catcgacacc
     1381 agcaaagtga attatggtgt aacggtactg ccgaccttca agggtcaacc atccaaaccg
     1441 ttcgttggcg tgctgagcgc aggtattaac gccgccagtc cgaacaaaga gctggcaaaa
     1501 gagttcctcg aaaactatct gctgactgat gaaggtctgg aagcggttaa taaagacaaa
     1561 ccgctgggtg ccgtagcgct gaagtcttac gaggaagagt tggcgaaaga tccacgtatt
     1621 gccgccacta tggaaaacgc ccagaaaggt gaaatcatgc cgaacatccc gcagatgtcc
     1681 gctttctggt atgccgtgcg tactgcggtg atcaacgccg ccagcggtcg tcagactgtc
     1741 gatgaagccc tgaaagacgc gcagactaat tcgagctcga acaacaacaa caataacaat
     1801 aacaacaacc tcgggatcga ggaaaacctg tacttccaat ccaatgcaat ggataagaaa
     1861 tactcaatag gcttagatat cggcacaaat agcgtcggat gggcggtgat cactgatgaa
     1921 tataaggttc cgtctaaaaa gttcaaggtt ctgggaaata cagaccgcca cagtatcaaa
     1981 aaaaatctta taggggctct tttatttgac agtggagaga cagcggaagc gactcgtctc
     2041 aaacggacag ctcgtagaag gtatacacgt cggaagaatc gtatttgtta tctacaggag
     2101 attttttcaa atgagatggc gaaagtagat gatagtttct ttcatcgact tgaagagtct
     2161 tttttggtgg aagaagacaa gaagcatgaa cgtcatccta tttttggaaa tatagtagat
     2221 gaagttgctt atcatgagaa atatccaact atctatcatc tgcgaaaaaa attggtagat
     2281 tctactgata aagcggattt gcgcttaatc tatttggcct tagcgcatat gattaagttt
     2341 cgtggtcatt ttttgattga gggagattta aatcctgata atagtgatgt ggacaaacta
     2401 tttatccagt tggtacaaac ctacaatcaa ttatttgaag aaaaccctat taacgcaagt
     2461 ggagtagatg ctaaagcgat tctttctgca cgattgagta aatcaagacg attagaaaat
     2521 ctcattgctc agctccccgg tgagaagaaa aatggcttat ttgggaatct cattgctttg
     2581 tcattgggtt tgacccctaa ttttaaatca aattttgatt tggcagaaga tgctaaatta
     2641 cagctttcaa aagatactta cgatgatgat ttagataatt tattggcgca aattggagat
     2701 caatatgctg atttgttttt ggcagctaag aatttatcag atgctatttt actttcagat
     2761 atcctaagag taaatactga aataactaag gctcccctat cagcttcaat gattaaacgc
     2821 tacgatgaac atcatcaaga cttgactctt ttaaaagctt tagttcgaca acaacttcca
     2881 gaaaagtata aagaaatctt ttttgatcaa tcaaaaaacg gatatgcagg ttatattgat
     2941 gggggagcta gccaagaaga attttataaa tttatcaaac caattttaga aaaaatggat
     3001 ggtactgagg aattattggt gaaactaaat cgtgaagatt tgctgcgcaa gcaacggacc
     3061 tttgacaacg gctctattcc ccatcaaatt cacttgggtg agctgcatgc tattttgaga
     3121 agacaagaag acttttatcc atttttaaaa gacaatcgtg agaagattga aaaaatcttg
     3181 acttttcgaa ttccttatta tgttggtcca ttggcgcgtg gcaatagtcg ttttgcatgg
     3241 atgactcgga agtctgaaga aacaattacc ccatggaatt ttgaagaagt tgtcgataaa
     3301 ggtgcttcag ctcaatcatt tattgaacgc atgacaaacg gcggtagtgt gtctggacaa
     3361 ggcgatagtt tacatgaaca tattgcaaat ttagctggta gccctgctat taaaaaaggt
     3421 attttacaga ctgtaaaagt tgttgatgaa ttggtcaaag taatggggcg gcataagcca
     3481 gaaaatatcg ttattgaaat ggcacgtgaa aatcagacaa ctcaaaaggg ccagaaaaat
     3541 tcgcgagagc gtatgaaacg aatcgaagaa ggtatcaaag aattaggaag tcagattctt
     3601 aaagagcatc ctgttgaaaa tactcaattg caaaatgaaa agctctatct ctattatctc
     3661 caaaatggaa gagacatgta tgtggaccaa gaattagata ttaatcgttt aagtgattat
     3721 gatgtcgatc acattgttcc acaaagtttc cttaaagacg attcaataga caataaggtc
     3781 ttaacgcgtt ctgataaaaa tcgtggtaaa tcggataacg ttccaagtga agaagtagtc
     3841 aaaaagatga aaaactattg gagacaactt ctaaacgcca agttaatcac tcaacgtaag
     3901 tttgataatt taacgaaagc tgaacgtgga ggtttgagtg aacttgataa agctggtttt
     3961 atcaaacgcc aattggttga aactcgccaa atcactaagc atgtggcaca aattttggat
     4021 agtcgcatga atactaaata cgatgaaaat gataaactta ttcgagaggt taaagtgatt
     4081 accttaaaat ctaaattagt ttctgacttc cgaaaagatt tccaattcta taaagtacgt
     4141 gagattaaca attaccatca tgcccatgat gcgtatctaa atgccgtcgt tggaactgct
     4201 ttgattaaga aatatccaaa acttgaatcg gagtttgtct atggtgatta taaagtttat
     4261 gatgttcgta aaatgattgc taagtctgag caagaaatag gcaaagcaac cgcaaaatat
     4321 ttcttttact ctaatatcat gaacttcttc aaaacagaaa ttacacttgc aaatggagag
     4381 attcgcaaac gccctctaat cgaaactaat ggggaaactg gagaaattgt ctgggataaa
     4441 gggcgagatt ttgccacagt gcgcaaagta ttgtccatgc cccaagtcaa tattgtcaag
     4501 aaaacagaag tacagacagg cggattctcc aaggagtcaa ttttaccaaa aagaaattcg
     4561 gacaagctta ttgctcgtaa aaaagactgg gatccaaaaa aatatggtgg ttttgatagt
     4621 ccaacggtag cttattcagt cctagtggtt gctaaggtgg aaaaagggaa atcgaagaag
     4681 ttaaaatccg ttaaagagtt actagggatc acaattatgg aaagaagttc ctttgaaaaa
     4741 aatccgattg actttttaga agctaaagga tataaggaag ttaaaaaaga cttaatcatt
     4801 aaactaccta aatatagtct ttttgagtta gaaaacggtc gtaaacggat gctggctagt
     4861 gccggagaat tacaaaaagg aaatgagctg gctctgccaa gcaaatatgt gaatttttta
     4921 tatttagcta gtcattatga aaagttgaag ggtagtccag aagataacga acaaaaacaa
     4981 ttgtttgtgg agcagcataa gcattattta gatgagatta ttgagcaaat cagtgaattt
     5041 tctaagcgtg ttattttagc agatgccaat ttagataaag ttcttagtgc atataacaaa
     5101 catagagaca aaccaatacg tgaacaagca gaaaatatta ttcatttatt tacgttgacg
     5161 aatcttggag ctcccgctgc ttttaaatat tttgatacaa caattgatcg taaacgatat
     5221 acgtctacaa aagaagtttt agatgccact cttatccatc aatccatcac tggtctttat
     5281 gaaacacgca ttgatttgag tcagctagga ggtgactaat aacattggaa gtggataacg
     5341 gatccgcgat cgcggcgcgc cacctggtgg ccggccggta ccacgcgtgc gcgctgatcc
     5401 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     5461 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     5521 tatatccgga tatccacagg acgggtgtgg tcgccatgat cgcgtagtcg atagtggctc
     5581 caagtagcga agcgagcagg actgggcggc ggccaaagcg gtcggacagt gctccgagaa
     5641 cgggtgcgca tagaaattgc atcaacgcat atagcgctag cagcacgcca tagtgactgg
     5701 cgatgctgtc ggaatggacg atatcccgca agaggcccgg cagtaccggc ataaccaagc
     5761 ctatgcctac agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt
     5821 tcatacacgg tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct
     5881 tatcgatgat aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc
     5941 ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt
     6001 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     6061 ccgctcatga gacaataacc ctgataaatg cttcaataac attgaaaaag gaagagtatg
     6121 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     6181 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     6241 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     6301 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     6361 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     6421 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     6481 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     6541 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     6601 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     6661 gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     6721 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6781 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6841 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6901 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6961 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     7021 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     7081 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     7141 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     7201 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     7261 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     7321 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     7381 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     7441 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     7501 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     7561 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     7621 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     7681 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     7741 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7801 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7861 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     7921 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     7981 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     8041 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     8101 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     8161 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     8221 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     8281 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     8341 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     8401 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     8461 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     8521 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     8581 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     8641 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     8701 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     8761 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     8821 tggccaggac ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc
     8881 gatggatatg ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt
     8941 ggctccaatt cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc
     9001 gaggtggccc ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg
     9061 gcgcctacaa tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg
     9121 acgatcagcg gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc
     9181 tgtccctgat ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg
     9241 atgccgccgg aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac
     9301 gccagcaaga cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg
     9361 ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg
     9421 aataccgcaa gcgacag
//
