LOCUS       sequence-198048-        6590 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for SpCas9 REC3 domain.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6590)
  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 6590)
  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: pSHS325 - Bacterial expression plasmid for SpC...
FEATURES             Location/Qualifiers
     source          1..6590
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     111..130
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     214..233
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        214..232
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             321..343
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             363..392
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             402..1502
                     /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     1476..1499
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1557..1577
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     primer_bind     complement(2333..2351)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2347..2394
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2760..2765,2766..2782,2783..2788))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2815..2833)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2901..3005
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3006..3074,3075..3866)
                     /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(3224..3243)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4037..4625
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4526..4545
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4779..4796
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4811..4950
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4876..4895)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5036..5058
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5052..5243)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
       61 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
      121 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
      181 gcgtagagga tcgagatctc gatcccgcga aattaatacg actcactata gggagaccac
      241 aacggtttcc ctctagtgcc ggctccggag agctctttaa ttaagcggcc gccctgcagg
      301 actcgagttc tagaaataat tttgtttaac tttaagaagg agatatacat atgaaatctt
      361 ctcaccatca ccatcaccat caccatcacc atggttcttc tatgaaaatc gaagaaggta
      421 aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa gtcggtaaga
      481 aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa ctggaagaga
      541 aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg gcacacgacc
      601 gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac aaagcgttcc
      661 aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag ctgattgctt
      721 acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg ccgaacccgc
      781 caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg
      841 cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct gctgacgggg
      901 gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc gtggataacg
      961 ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa cacatgaatg
     1021 cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca gcgatgacca
     1081 tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat ggtgtaacgg
     1141 tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg agcgcaggta
     1201 ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac tatctgctga
     1261 ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta gcgctgaagt
     1321 cttacgagga agagttggcg aaagatccac gtattgccgc cactatggaa aacgcccaga
     1381 aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc gtgcgtactg
     1441 cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa gacgcgcaga
     1501 ctaattcgag ctcgaacaac aacaacaata acaataacaa caacctcggg atcgaggaaa
     1561 acctgtactt ccaatccaat gcaaaagtac taccaaaaca tagtttgctt tatgagtatt
     1621 ttacggttta taacgaattg acaaaggtca aatatgttac tgaaggaatg cgaaaaccag
     1681 catttctttc aggtgaacag aagaaagcca ttgttgattt actcttcaaa acaaatcgaa
     1741 aagtaaccgt taagcaatta aaagaagatt atttcaaaaa aatagaatgt tttgatagtg
     1801 ttgaaatttc aggagttgaa gatagattta atgcttcatt aggtacctac catgatttgc
     1861 taaaaattat taaagataaa gattttttgg ataatgaaga aaatgaagat atcttagagg
     1921 atattgtttt aacattgacc ttatttgaag atagggagat gattgaggaa agacttaaaa
     1981 catatgctca cctctttgat gataaggtga tgaaacagct taaacgtcgc cgttatactg
     2041 gttggggacg tttgtctcga aaattgatta atggtattag ggataagcaa tctggcaaaa
     2101 caatattaga ttttttgaaa tcagatggtt ttgccaatcg caattttatg cagctgatcc
     2161 atgatgatag tttgacattt aaagaagaca ttcaaaaagc acaataataa cattggaagt
     2221 ggataacgga tccgcgatcg cggcgcgcca cctggtggcc ggccggtacc acgcgtgcgc
     2281 gctgatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     2341 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     2401 ggaggaacta tatccggata tccacaggac gggtgtggtc gccatgatcg cgtagtcgat
     2461 agtggctcca agtagcgaag cgagcaggac tgggcggcgg ccaaagcggt cggacagtgc
     2521 tccgagaacg ggtgcgcata gaaattgcat caacgcatat agcgctagca gcacgccata
     2581 gtgactggcg atgctgtcgg aatggacgat atcccgcaag aggcccggca gtaccggcat
     2641 aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt
     2701 gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca
     2761 ttaaagctta tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg
     2821 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     2881 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2941 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataacat tgaaaaagga
     3001 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     3061 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     3121 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     3181 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     3241 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     3301 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     3361 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     3421 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     3481 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     3541 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     3601 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     3661 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3721 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     3781 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3841 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     3901 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     3961 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4021 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     4081 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     4141 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     4201 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     4261 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     4321 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     4381 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     4441 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     4501 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     4561 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     4621 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     4681 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4741 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4801 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4861 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     4921 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     4981 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     5041 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     5101 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     5161 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     5221 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     5281 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     5341 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     5401 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     5461 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     5521 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     5581 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     5641 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     5701 cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg
     5761 gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag
     5821 aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca
     5881 ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt
     5941 atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa
     6001 tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc
     6061 cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg
     6121 gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg
     6181 tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct
     6241 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     6301 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     6361 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     6421 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     6481 tgaaggctct caagggcatc ggtcgacgct ctcccttatg cgactcctgc attaggaagc
     6541 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
//
