LOCUS       sequence-247953-        3696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cloning vector FnoCas12a DR-crRNA for expression in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3696)
  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 3696)
  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: FnoCas12a-DR-cr in PBCSK-
FEATURES             Location/Qualifiers
     source          1..3696
                     /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(1069..1089)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1069..1087)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1108..1124)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1108..1124)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1121..1143)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1132..1148
                     /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(1156..1162,1163..1180,1181..1186))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1201..1222
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1339..1356)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1510..2098)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1590..1609)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2318..2420
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             2421..3080
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2487..2506)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3569..3673)
                     /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 accgtctaag aactttaaat aatttctact gttgtagatt tgtcttcgaa
     1021 gacaattttt ttgcggccgc caccgcggtg gagctccagc ttttgttccc tttagtgagg
     1081 gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc
     1141 gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     1201 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     1261 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     1321 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     1381 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     1441 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     1501 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     1561 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     1621 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     1681 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     1741 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     1801 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     1861 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     1921 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     1981 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     2041 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     2101 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     2161 gattttggtc atgagattat caaaaaggat cttcacctag atccttttcg accgaataaa
     2221 tacctgtgac ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg
     2281 ggaagccctg ggccaacttt tggcgaaaat gagacgttga tcggcacgta agaggttcca
     2341 actttcacca taatgaaata agatcactac cgggcgtatt ttttgagttg tcgagatttt
     2401 caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat
     2461 cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata
     2521 accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca
     2581 agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaattac
     2641 gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg
     2701 ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc
     2761 ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt
     2821 tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca
     2881 ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg
     2941 gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg
     3001 ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg
     3061 agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa acgcctggtt
     3121 gctacgcctg aataagtgat aataagcgga tgaatggcag aaattcgaaa gcaaattcga
     3181 cccggtcgtc ggttcagggc agggtcgtta aatagccgct tatgtctatt gctggtttac
     3241 cggtttattg actaccggaa gcagtgtgac cgtgtgcttc tcaaatgcct gaggccagtt
     3301 tgctcaggct ctccccgtgg aggtaataat tgacgatatg atcctttttt tctgatcaaa
     3361 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3421 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3481 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3541 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3601 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3661 taggggttcc gcgcacattt ccccgaaaag tgccac
//
