LOCUS       sequence-247924-        3695 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector AsCas12a DR-crRNA for expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3695)
  AUTHORS   Liu P, Luk K, Shin M, Idrizi F, Kwok S, Roscoe B, Mintzer E, Suresh 
            S, Morrison K, Frazao JB, Bolukbasi MF, Ponnienselvan K, Luban J, 
            Zhu LJ, Lawson ND, Wolfe SA
  TITLE     Enhanced Cas12a editing in mammalian cells and zebrafish.
  JOURNAL   Nucleic Acids Res. 2019 Mar 20. pii: 5403491. doi: 
            10.1093/nar/gkz184.
  PUBMED    30892626
REFERENCE   2  (bases 1 to 3695)
  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: "Nucleic 
            Acids Res. 2019 Mar 20. pii: 5403491. doi: 10.1093/nar/gkz184."
COMMENT     Sequence Label: AsCas12a-DR-cr in PBCSK-
FEATURES             Location/Qualifiers
     source          1..3695
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     669..685
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        725..973
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     725..745
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     896..915
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1068..1088)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1068..1086)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1107..1123)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1107..1123)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1120..1142)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1131..1147
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1155..1161,1162..1179,1180..1185))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1200..1221
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1338..1355)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1509..2097)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1589..1608)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2317..2419
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             2420..3079
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2486..2505)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3568..3672)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc agcccggggg
      721 atccgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
      781 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
      841 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
      901 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
      961 aggacgaaac accgtcaaaa gaccttttta atttctactc ttgtagattt gtcttcgaag
     1021 acaatttttt tgcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg
     1081 ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     1141 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     1201 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     1261 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     1321 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     1381 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     1441 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     1501 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     1561 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     1621 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     1681 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     1741 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     1801 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     1861 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     1921 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     1981 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     2041 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     2101 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     2161 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttcga ccgaataaat
     2221 acctgtgacg gaagatcact tcgcagaata aataaatcct ggtgtccctg ttgataccgg
     2281 gaagccctgg gccaactttt ggcgaaaatg agacgttgat cggcacgtaa gaggttccaa
     2341 ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttgt cgagattttc
     2401 aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg ttgatatatc
     2461 ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat gtacctataa
     2521 ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa ataagcacaa
     2581 gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc cggaattacg
     2641 tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt gttacaccgt
     2701 tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg acgatttccg
     2761 gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc tggcctattt
     2821 ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg tgagtttcac
     2881 cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt tcaccatggg
     2941 caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg ttcatcatgc
     3001 cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt actgcgatga
     3061 gtggcagggc ggggcgtaat ttttttaagg cagttattgg tgcccttaaa cgcctggttg
     3121 ctacgcctga ataagtgata ataagcggat gaatggcaga aattcgaaag caaattcgac
     3181 ccggtcgtcg gttcagggca gggtcgttaa atagccgctt atgtctattg ctggtttacc
     3241 ggtttattga ctaccggaag cagtgtgacc gtgtgcttct caaatgcctg aggccagttt
     3301 gctcaggctc tccccgtgga ggtaataatt gacgatatga tccttttttt ctgatcaaaa
     3361 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     3421 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     3481 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     3541 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     3601 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     3661 aggggttccg cgcacatttc cccgaaaagt gccac
//
