LOCUS       sequence-203059-        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        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    416..533
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        582..1092
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1011..1029
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1114..1713
                     /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(1114..1133)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1610..1630
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    1860..1981
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1871..1890)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1925..1944
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2229..2246)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2400..2988)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2480..2499)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3159..3950,3951..4019))
                     /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     3782..3801
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4020..4124)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4151..4606
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4238..4257)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4448..4469
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    4737..4785
                     /note="synthetic polyadenylation signal"
     misc_feature    4799..4890
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     4839..4858
                     /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 taagtgtaga tcgagacccg agagagggtc
      361 ggtctccttt ttttgaattc tcgacctcga gacaaatggc agtattcatc cacaatttta
      421 aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa
      481 cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt
      541 attacaggga cagcagagat ccactttggc cgcggctcga gggggttggg gttgcgcctt
      601 ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa
      661 cgcagcggcg ccgaccctgg gactcgcaca ttcttcacgt ccgttcgcag cgtcacccgg
      721 atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg
      781 gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact
      841 agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct
      901 gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg
      961 cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca
     1021 ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac
     1081 ctctctcccc agggggatcc accggagctt accatgaccg agtacaagcc cacggtgcgc
     1141 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     1201 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     1261 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     1321 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     1381 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     1441 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     1501 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     1561 gcggccgagc gcgccggggt gcccgccttc ctggaaacct ccgcgccccg caacctcccc
     1621 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     1681 acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg
     1741 accgaaagga gcgcacgacc ccatgcatcg gtacctttaa gaccaatgac ttacaaggca
     1801 gctgtagatc ttagccactt tctagagtcg gggcggccgg ccgcttcgag cagacatgat
     1861 aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa aatgctttat
     1921 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     1981 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     2041 ttaaagcaag taaaacctct acaaatgtgg taaaatcgat aaggatccgt cgaccgatgc
     2101 ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg
     2161 ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
     2221 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     2281 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     2341 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     2401 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     2461 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     2521 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     2581 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     2641 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     2701 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     2761 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     2821 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     2881 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     2941 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     3001 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     3061 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     3121 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     3181 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     3241 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcgg
     3301 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     3361 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     3421 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     3481 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     3541 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     3601 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     3661 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     3721 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     3781 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     3841 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     3901 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     3961 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     4021 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     4081 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     4141 gtgccacctg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     4201 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4261 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4321 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     4381 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4441 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     4501 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     4561 caaaaattta acgcgaattt taacaaaata ttaacgttta caatttccca ttcgccattc
     4621 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagccc
     4681 aagctaccat gataagtaag taatattaag gtacgggagg tacttggagc ggccgcaata
     4741 aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgaatcg atagtactaa
     4801 catacgctct ccatcaaaac aaaacgaaac aaaacaaact agcaaaatag gctgtcccca
     4861 gtgcaagtgc aggtgccaga acatttctct atcgata
//
