LOCUS       sequence-207566-        4897 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses LbCpf1 crRNA in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4897)
  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 4897)
  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: pLb-Cpf1-pGL3-U6-sgRNA
FEATURES             Location/Qualifiers
     source          1..4897
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        61..309
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     61..81
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     232..251
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    405..522
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        571..1081
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1000..1018
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1103..1702
                     /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(1103..1122)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1599..1619
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1849..1970
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1860..1879)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1914..1933
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2218..2235)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2389..2977)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2469..2488)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3148..3939,3940..4008))
                     /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     3771..3790
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4009..4113)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4140..4595
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4227..4246)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4437..4458
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4726..4774
                     /note="synthetic polyadenylation signal"
     misc_feature    4788..4879
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4828..4847
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 gtgaacggat ctcgacggta tcgatcacga gactagcctc gagcggccgc ccccttcacc
       61 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
      121 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      181 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      241 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      301 cgaaacaccg aatttctact aagtgtagat cgagacccga gagagggtcg gtctcctttt
      361 tttgaattct cgacctcgag acaaatggca gtattcatcc acaattttaa aagaaaaggg
      421 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
      481 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
      541 agcagagatc cactttggcc gcggctcgag ggggttgggg ttgcgccttt tccaaggcag
      601 ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc
      661 cgaccctggg actcgcacat tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc
      721 tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt
      781 gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc
      841 agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg tggccaatag
      901 cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc gggaggcggg
      961 gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc
     1021 tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc tctctcccca
     1081 gggggatcca ccggagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg
     1141 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     1201 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     1261 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     1321 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     1381 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     1441 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga
     1501 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     1561 cgccggggtg cccgccttcc tggaaacctc cgcgccccgc aacctcccct tctacgagcg
     1621 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     1681 gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag
     1741 cgcacgaccc catgcatcgg tacctttaag accaatgact tacaaggcag ctgtagatct
     1801 tagccacttt ctagagtcgg ggcggccggc cgcttcgagc agacatgata agatacattg
     1861 atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt
     1921 gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca
     1981 attgcattca ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt
     2041 aaaacctcta caaatgtggt aaaatcgata aggatccgtc gaccgatgcc cttgagagcc
     2101 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
     2161 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctt ccgcttcctc
     2221 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     2281 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     2341 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     2401 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     2461 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     2521 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     2581 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     2641 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     2701 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     2761 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     2821 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     2881 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     2941 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     3001 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     3061 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     3121 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     3181 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     3241 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcggg acccacgctc
     3301 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     3361 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     3421 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     3481 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     3541 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     3601 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     3661 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     3721 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     3781 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     3841 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     3901 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     3961 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     4021 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     4081 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     4141 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4201 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4261 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4321 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     4381 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4441 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4501 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4561 cgcgaatttt aacaaaatat taacgcttac aatttgccat tcgccattca ggctgcgcaa
     4621 ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagccca agctaccatg
     4681 ataagtaagt aatattaagg tacgggaggt acttggagcg gccgcaataa aatatcttta
     4741 ttttcattac atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc
     4801 catcaaaaca aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca
     4861 ggtgccagaa catttctcta tcgataggta ccgatta
//
