LOCUS       sequence-478979-        4892 bp    DNA     circular SYN 17-JUL-2025
DEFINITION  Overexpression of HyperdLbCas12a in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4892)
  AUTHORS   Yang LZ, Min YH, Liu YX, Gao BQ, Liu XQ, Huang Y, Wang H, Yang L, 
            Liu ZJ, Chen LL
  TITLE     CRISPR-array-mediated imaging of non-repetitive and multiplex 
            genomic loci in living cells.
  JOURNAL   Nat Methods. 2024 Sep;21(9):1646-1657. doi: 
            10.1038/s41592-024-02333-3. Epub 2024 Jul 4.
  PUBMED    38965442
REFERENCE   2  (bases 1 to 4892)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1038/s41592-024-02333-3"; journalName: "Nat Methods"; date: 
            "2024-09"; volume: "21"; issue: "9"; pages: "1646-1657"
COMMENT     Sequence Label: pHAGE-IRES-puro-NLS-HyperdLbCas12a-tagBFP-NLS-...
FEATURES             Location/Qualifiers
     source          1..4892
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     364..383
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(602..706)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      733..1188
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(820..839)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1030..1051
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    1319..1367
                     /note="synthetic polyadenylation signal"
     misc_feature    1381..1472
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     1421..1440
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     promoter        1551..1799
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1551..1571
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1722..1741
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    1890..2007
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2056..2566
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2485..2503
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2588..3187
                     /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(2588..2607)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3084..3104
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    3334..3455
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3345..3364)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3399..3418
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3703..3720)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3874..4462)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3954..3973)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4633..4892,1..532,533..601))
                     /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"
ORIGIN
        1 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
       61 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
      121 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
      181 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
      241 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
      301 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
      361 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
      421 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
      481 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
      541 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
      601 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
      661 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
      721 aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
      781 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
      841 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
      901 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
      961 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     1021 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     1081 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     1141 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttgc cattcgccat
     1201 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     1261 ccaagctacc atgataagta agtaatatta aggtacggga ggtacttgga gcggccgcaa
     1321 taaaatatct ttattttcat tacatctgtg tgttggtttt ttgtgtgaat cgatagtact
     1381 aacatacgct ctccatcaaa acaaaacgaa acaaaacaaa ctagcaaaat aggctgtccc
     1441 cagtgcaagt gcaggtgcca gaacatttct ctatcgatag gtaccgatta gtgaacggat
     1501 ctcgacggta tcgatcacga gactagcctc gagcggccgc ccccttcacc gagggcctat
     1561 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     1621 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     1681 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     1741 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     1801 taatttctac taagtgtaga tggaggccga ggcgggcgga ttttttttga attctcgacc
     1861 tcgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac
     1921 agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa
     1981 aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt
     2041 tggccgcggc tcgagggggt tggggttgcg ccttttccaa ggcagccctg ggtttgcgca
     2101 gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc ctgggactcg
     2161 cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc
     2221 cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg
     2281 ccggacgtga caaacggaag ccgcacgtct cactagtacc ctcgcagacg gacagcgcca
     2341 gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct gctcagcagg
     2401 gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt
     2461 gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc
     2521 ggcagtcggc tccctcgttg accgaatcac cgacctctct ccccaggggg atccaccgga
     2581 gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag
     2641 ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga
     2701 tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg
     2761 gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac
     2821 gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt
     2881 gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc
     2941 caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc agggcaaggg
     3001 tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc
     3061 cttcctggaa acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt
     3121 caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg
     3181 tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgc
     3241 atcggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttctaga
     3301 gtcggggcgg ccggccgctt cgagcagaca tgataagata cattgatgag tttggacaaa
     3361 ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt
     3421 tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta
     3481 tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat
     3541 gtggtaaaat cgataaggat ccgtcgaccg atgcccttga gagccttcaa cccagtcagc
     3601 tccttccggt gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc
     3661 atgcaactcg taggacaggt gccggcagcg ctcttccgct tcctcgctca ctgactcgct
     3721 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3781 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3841 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3901 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3961 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4021 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4081 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4141 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4201 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4261 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4321 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4381 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4441 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4501 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4561 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4621 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4681 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4741 accatctggc cccagtgctg caatgatacc gcgggaccca cgctcaccgg ctccagattt
     4801 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4861 cgcctccatc cagtctatta attgttgccg gg
//
