LOCUS       sequence-346768-        3636 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SpCas9 guide cloning vector derived from BPK1520 that allows for 
            puromycin-based selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3636)
  AUTHORS   Erwood S, Brewer RA, Bily TMI, Maino E, Zhou L, Cohn RD, Ivakine EA
  TITLE     Modeling Niemann-Pick disease type C in a human haploid cell line 
            allows for patient variant characterization and clinical 
            interpretation.
  JOURNAL   Genome Res. 2019 Dec;29(12):2010-2019. doi: 10.1101/gr.250720.119. 
            Epub 2019 Nov 21.
  PUBMED    31754021
REFERENCE   2  (bases 1 to 3636)
  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"; doi: 
            "10.1101/gr.250720.119"; journalName: "Genome Res"; date: "2019-12";
            volume: "29"; issue: "12"; pages: "2010-2019"
COMMENT     Sequence Label: BPK1520_puroR
FEATURES             Location/Qualifiers
     source          1..3636
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        41..420
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        421..624
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     575..595
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             649..1248
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(649..668)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1145..1165
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1274..1395
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1311..1330)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1365..1384
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1438..1686
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1438..1458
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1609..1628
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1708..1783
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(1887..2475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1967..1986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2646..3437,3438..3506))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3269..3288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3507..3611)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgcta gctctagagg gcctgaattc tcgaggcgtt gacattgatt attgactagt
       61 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
      121 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
      181 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
      241 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
      301 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
      361 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
      421 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
      481 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      541 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      601 tgggaggtct atataagcag agctctctgg ctaactaccg gtgccaccat gaccgagtac
      661 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
      721 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
      781 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
      841 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
      901 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
      961 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
     1021 ctggccaccg tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
     1081 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     1141 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     1201 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaga attcatcgat
     1261 accgtcgaga tctaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     1321 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     1381 catcaatgta tcttatcatg tctggaaatt cgatccggta ccaaggtcgg gcaggaagag
     1441 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
     1501 attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
     1561 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
     1621 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
     1681 aacaccggag acgattaatg cgtctccgtt ttagagctag aaatagcaag ttaaaataag
     1741 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt aagcttgggc
     1801 cgctcgaggt acctctctac atatgacatg tgagcaaaag gccagcaaaa ggccaggaac
     1861 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     1921 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     1981 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2041 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     2101 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     2161 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     2221 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     2281 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     2341 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     2401 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     2461 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     2521 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     2581 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     2641 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     2701 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     2761 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     2821 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     2881 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     2941 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3001 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3061 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     3121 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     3181 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     3241 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     3301 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     3361 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     3421 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     3481 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     3541 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     3601 ggggttccgc gcacatttcc ccgaaaagtg ccacct
//
