LOCUS       sequence-203060-        4896 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses AsCpf1 crRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4896)
  AUTHORS   Li X, Wang Y, Liu Y, Yang B, Wang X, Wei J, Lu Z, Zhang Y, Wu J, 
            Huang X, Yang L, Chen J
  TITLE     Base editing with a Cpf1-cytidine deaminase fusion.
  JOURNAL   Nat Biotechnol. 2018 Mar 19. pii: nbt.4102. doi: 10.1038/nbt.4102.
  PUBMED    29553573
REFERENCE   2  (bases 1 to 4896)
  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: "Nat 
            Biotechnol. 2018 Mar 19. pii: nbt.4102. doi: 10.1038/nbt.4102."
COMMENT     Sequence Label: pAs-Cpf1-pGL3-U6-sgRNA
FEATURES             Location/Qualifiers
     source          1..4896
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        72..320
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     72..92
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     243..262
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    415..532
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        581..1091
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1010..1028
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1113..1712
                     /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(1113..1132)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1609..1629
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1859..1980
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1870..1889)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1924..1943
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2228..2245)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2399..2987)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2479..2498)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3158..3949,3950..4018))
                     /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     3781..3800
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4019..4123)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4150..4605
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4237..4256)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4447..4468
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4736..4784
                     /note="synthetic polyadenylation signal"
     misc_feature    4798..4889
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4838..4857
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
       61 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
      121 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
      181 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
      241 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
      301 tgtggaaagg acgaaacacc gaatttctac tcttgtagat cgagacccga gagagggtcg
      361 gtctcctttt tttgaattct cgacctcgag acaaatggca gtattcatcc acaattttaa
      421 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
      481 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
      541 ttacagggac agcagagatc cactttggcc gcggctcgag ggggttgggg ttgcgccttt
      601 tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac
      661 gcagcggcgc cgaccctggg actcgcacat tcttcacgtc cgttcgcagc gtcacccgga
      721 tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg
      781 aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta
      841 gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg
      901 tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc
      961 gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat
     1021 tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc
     1081 tctctcccca gggggatcca ccggagctta ccatgaccga gtacaagccc acggtgcgcc
     1141 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     1201 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     1261 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     1321 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     1381 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     1441 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     1501 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     1561 cggccgagcg cgccggggtg cccgccttcc tggaaacctc cgcgccccgc aacctcccct
     1621 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     1681 cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga
     1741 ccgaaaggag cgcacgaccc catgcatcgg tacctttaag accaatgact tacaaggcag
     1801 ctgtagatct tagccacttt ctagagtcgg ggcggccggc cgcttcgagc agacatgata
     1861 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt
     1921 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt
     1981 aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt
     2041 taaagcaagt aaaacctcta caaatgtggt aaaatcgata aggatccgtc gaccgatgcc
     2101 cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc
     2161 cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctt
     2221 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     2281 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     2341 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     2401 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     2461 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     2521 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     2581 tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     2641 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     2701 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     2761 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     2821 actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
     2881 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     2941 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3001 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     3061 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     3121 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     3181 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     3241 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcggg
     3301 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     3361 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     3421 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     3481 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     3541 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     3601 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     3661 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     3721 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     3781 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     3841 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     3901 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     3961 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4021 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     4081 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     4141 tgccacctga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4201 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4261 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4321 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     4381 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4441 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     4501 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     4561 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aatttcccat tcgccattca
     4621 ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagccca
     4681 agctaccatg ataagtaagt aatattaagg tacgggaggt acttggagcg gccgcaataa
     4741 aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagtactaac
     4801 atacgctctc catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag
     4861 tgcaagtgca ggtgccagaa catttctcta tcgata
//
