LOCUS       sequence-197572-        9437 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for SpCas9∆REC3, HNH 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 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: pJSC038 - Bacterial expression plasmid for
            SpC...
FEATURES             Location/Qualifiers
     source          1..9437
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     462..481
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     565..584
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        565..583
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             672..694
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             714..743
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             753..1853
                     /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     1827..1850
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1908..1928
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(5531..5549)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5545..5592
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(5958..5963,5964..5980,5981..5986))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(6013..6031)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6099..6203
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6204..6272,6273..7064)
                     /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(6422..6441)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7235..7823
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7724..7743
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7977..7994
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    8009..8148
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(8074..8093)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8234..8256
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(8250..8441)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc
       61 aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc
      121 tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag
      181 acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg
      241 ttgaaggctc tcaagggcat cggtcgacgc tctcccttat gcgactcctg cattaggaag
      301 cagcccagta gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag
      361 gagatggcgc ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa
      421 gcgctcatga gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag
      481 gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg
      541 atcgagatct cgatcccgcg aaattaatac gactcactat agggagacca caacggtttc
      601 cctctagtgc cggctccgga gagctcttta attaagcggc cgccctgcag gactcgagtt
      661 ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgaaatct tctcaccatc
      721 accatcacca tcaccatcac catggttctt ctatgaaaat cgaagaaggt aaactggtaa
      781 tctggattaa cggcgataaa ggctataacg gtctcgctga agtcggtaag aaattcgaga
      841 aagataccgg aattaaagtc accgttgagc atccggataa actggaagag aaattcccac
      901 aggttgcggc aactggcgat ggccctgaca ttatcttctg ggcacacgac cgctttggtg
      961 gctacgctca atctggcctg ttggctgaaa tcaccccgga caaagcgttc caggacaagc
     1021 tgtatccgtt tacctgggat gccgtacgtt acaacggcaa gctgattgct tacccgatcg
     1081 ctgttgaagc gttatcgctg atttataaca aagatctgct gccgaacccg ccaaaaacct
     1141 gggaagagat cccggcgctg gataaagaac tgaaagcgaa aggtaagagc gcgctgatgt
     1201 tcaacctgca agaaccgtac ttcacctggc cgctgattgc tgctgacggg ggttatgcgt
     1261 tcaagtatga aaacggcaag tacgacatta aagacgtggg cgtggataac gctggcgcaa
     1321 aagcgggtct gaccttcctg gttgacctga ttaaaaacaa acacatgaat gcagacaccg
     1381 attactccat cgcagaagct gcctttaata aaggcgaaac agcgatgacc atcaacggcc
     1441 cgtgggcatg gtccaacatc gacaccagca aagtgaatta tggtgtaacg gtactgccga
     1501 ccttcaaggg tcaaccatcc aaaccgttcg ttggcgtgct gagcgcaggt attaacgccg
     1561 ccagtccgaa caaagagctg gcaaaagagt tcctcgaaaa ctatctgctg actgatgaag
     1621 gtctggaagc ggttaataaa gacaaaccgc tgggtgccgt agcgctgaag tcttacgagg
     1681 aagagttggc gaaagatcca cgtattgccg ccactatgga aaacgcccag aaaggtgaaa
     1741 tcatgccgaa catcccgcag atgtccgctt tctggtatgc cgtgcgtact gcggtgatca
     1801 acgccgccag cggtcgtcag actgtcgatg aagccctgaa agacgcgcag actaattcga
     1861 gctcgaacaa caacaacaat aacaataaca acaacctcgg gatcgaggaa aacctgtact
     1921 tccaatccaa tgcaatggat aagaaatact caataggctt agatatcggc acaaatagcg
     1981 tcggatgggc ggtgatcact gatgaatata aggttccgtc taaaaagttc aaggttctgg
     2041 gaaatacaga ccgccacagt atcaaaaaaa atcttatagg ggctctttta tttgacagtg
     2101 gagagacagc ggaagcgact cgtctcaaac ggacagctcg tagaaggtat acacgtcgga
     2161 agaatcgtat ttcttatcta caggagattt tttcaaatga gatggcgaaa gtagatgata
     2221 gtttctttca tcgacttgaa gagtcttttt tggtggaaga agacaagaag catgaacgtc
     2281 atcctatttt tggaaatata gtagatgaag ttgcttatca tgagaaatat ccaactatct
     2341 atcatctgcg aaaaaaattg gtagattcta ctgataaagc ggatttgcgc ttaatctatt
     2401 tggccttagc gcatatgatt aagtttcgtg gtcatttttt gattgaggga gatttaaatc
     2461 ctgataatag tgatgtggac aaactattta tccagttggt acaaacctac aatcaattat
     2521 ttgaagaaaa ccctattaac gcaagtggag tagatgctaa agcgattctt tctgcacgat
     2581 tgagtaaatc aagacgatta gaaaatctca ttgctcagct ccccggtgag aagaaaaatg
     2641 gcttatttgg gaatctcatt gctttgtcat tgggtttgac ccctaatttt aaatcaaatt
     2701 ttgatttggc agaagatgct aaattacagc tttcaaaaga tacttacgat gatgatttag
     2761 ataatttatt ggcgcaaatt ggagatcaat atgctgattt gtttttggca gctaagaatt
     2821 tatcagatgc tattttactt tcagatatcc taagagtaaa tactgaaata actaaggctc
     2881 ccctatcagc ttcaatgatt aaacgctacg atgaacatca tcaagacttg actcttttaa
     2941 aagctttagt tcgacaacaa cttccagaaa agtataaaga aatctttttt gatcaatgta
     3001 aaaacggata tgcaggttat attgatgggg gagctagcca agaagaattt tataaattta
     3061 tcaaaccaat tttagaaaaa atggatggta ctgaggaatt attggtgaaa ctaaatcgtg
     3121 aagatttgct gcgcaagcaa cggacctttg acaacggctc tattccccat caaattcact
     3181 tgggtgagct gcatgctatt ttgagaagac aagaagactt ttatccattt ttaaaagaca
     3241 atcgtgagaa gattgaaaaa atcttgactt ttcgaattcc ttattatgtt ggtccattgg
     3301 cgcgtggcaa tagtcgtttt gcatggatga ctcggaagtc tgaagaaaca attaccccat
     3361 ggaattttga agaagttgtc gataaaggtg cttcagctca atcatttatt gaacgcatga
     3421 caaacggtgg aagtgtgtct ggacaaggcg atagtttaca tgaacatatt gcaaatttag
     3481 ctggtagccc tgctattaaa aaaggtattt tacagactgt aaaagttgtt gatgaattgg
     3541 tcaaagtaat ggggcggcat aagccagaaa atatagttat tgaaatggca cgtgaaaatc
     3601 agacaactca aaagggccag aaaaattcgc gagagcgtat gaaacgaatc gaagaaggta
     3661 tcaaagaatt aggaagtcag attcttaaag agcatcctgt tgaaaatact caattgcaaa
     3721 atgaaaagct ctatctctat tatctccaaa atggaagaga catgtatgtg gaccaagaat
     3781 tagatattaa tcgtttaagt gattatgatg tcgatcacat tgttccacaa agtttcctta
     3841 aagacgattc aatagacaat aaggtcttaa cgcgttctga taaaaatcgt ggtaaatgcg
     3901 ataacgttcc aagtgaagaa gtagtcaaaa agatgaaaaa ctattggaga caacttctaa
     3961 acgccaagtt aatcactcaa cgtaagtttg ataatttaac gaaagctgaa cgtggaggtt
     4021 tgagtgaact tgataaagct ggttttatca aacgccaatt ggttgaaact cgccaaatca
     4081 ctaagcatgt ggcacaaatt ttggatagtc gcatgaatac taaatacgat gaaaatgata
     4141 aacttattcg agaggttaaa gtgattacct taaaatctaa attagtttct gacttccgaa
     4201 aagatttcca attctataaa gtacgtgaga ttaacaatta ccatcatgcc catgatgcgt
     4261 atctaaatgc cgtcgttgga actgctttga ttaagaaata tccaaaactt gaatcggagt
     4321 ttgtctatgg tgattataaa gtttatgatg ttcgtaaaat gattgctaag tctgagcaag
     4381 aaataggcaa agcaaccgca aaatatttct tttactctaa tatcatgaac ttcttcaaaa
     4441 cagaaattac acttgcaaat ggagagattc gcaaacgccc tctaatcgaa actaatgggg
     4501 aaactggaga aattgtctgg gataaagggc gagattttgc cacagtgcgc aaagtattgt
     4561 ccatgcccca agtcaatatt gtcaagaaaa cagaagtaca gacaggcgga ttctccaagg
     4621 agtcaatttt accaaaaaga aattcggaca agcttattgc tcgtaaaaaa gactgggatc
     4681 caaaaaaata tggtggtttt gatagtccaa cggtagctta ttcagtccta gtggttgcta
     4741 aggtggaaaa agggaaatcg aagaagttaa aatccgttaa agagttacta gggatcacaa
     4801 ttatggaaag aagttccttt gaaaaaaatc cgattgactt tttagaagct aaaggatata
     4861 aggaagttaa aaaagactta atcattaaac tacctaaata tagtcttttt gagttagaaa
     4921 acggtcgtaa acggatgctg gctagtgccg gagaattaca aaaaggaaat gagctggctc
     4981 tgccaagcaa atatgtgaat tttttatatt tagctagtca ttatgaaaag ttgaagggta
     5041 gtccagaaga taacgaacaa aaacaattgt ttgtggagca gcataagcat tatttagatg
     5101 agattattga gcaaatcagt gaattttcta agcgtgttat tttagcagat gccaatttag
     5161 ataaagttct tagtgcatat aacaaacata gagacaaacc aatacgtgaa caagcagaaa
     5221 atattattca tttatttacg ttgacgaatc ttggagctcc cgctgctttt aaatattttg
     5281 atacaacaat tgatcgtaaa cgatatacgt ctacaaaaga agttttagat gccactctta
     5341 tccatcaatc catcactggt ctttatgaaa cacgcattga tttgagtcag ctaggaggtg
     5401 actaataaca ttggaagtgg ataacggatc cgcgatcgcg gcgcgccacc tggtggccgg
     5461 ccggtaccac gcgtgcgctc tgatccggct gctaacaaag cccgaaagga agctgagttg
     5521 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     5581 aggggttttt tgctgaaagg aggaactata tccggatatc cacaggacgg gtgtggtcgc
     5641 catgatcgcg tagtcgatag tggctccaag tagcgaagcg agcaggactg ggcggcggcc
     5701 aaagcggtcg gacagtgctc cgagaacggg tgcgcataga aattgcatca acgcatatag
     5761 cgctagcagc acgccatagt gactggcgat gctgtcggaa tggacgatat cccgcaagag
     5821 gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag
     5881 gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa
     5941 ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt
     6001 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     6061 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     6121 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     6181 aataacattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     6241 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     6301 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     6361 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     6421 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     6481 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     6541 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     6601 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     6661 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     6721 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     6781 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     6841 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     6901 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     6961 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     7021 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     7081 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     7141 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     7201 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     7261 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     7321 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     7381 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     7441 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     7501 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     7561 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     7621 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     7681 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     7741 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     7801 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     7861 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     7921 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     7981 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     8041 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     8101 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     8161 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     8221 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     8281 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     8341 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     8401 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     8461 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     8521 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     8581 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     8641 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     8701 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     8761 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     8821 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     8881 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc
     8941 gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca
     9001 ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg
     9061 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg
     9121 cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc
     9181 tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg
     9241 taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca
     9301 gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga
     9361 aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     9421 tgccggcgat aatggcc
//
