LOCUS       sequence-228915-        3702 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FnCas12a-gRNA targeting TLR2.0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3702)
  AUTHORS   Iyer S, Mir A, Vega-Badillo J, Roscoe BP, Ibraheim R, Zhu LJ, Lee J,
            Liu P, Luk K, Mintzer E, Guo D, Soares de Brito J, Emerson CP Jr, 
            Zamore PD, Sontheimer EJ, Wolfe SA
  TITLE     Efficient Homology-Directed Repair with Circular Single-Stranded DNA
            Donors.
  JOURNAL   CRISPR J. 2022 Oct;5(5):685-701. doi: 10.1089/crispr.2022.0058. Epub
            2022 Sep 7.
  PUBMED    36070530
REFERENCE   2  (bases 1 to 3702)
  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"; doi: 
            "10.1089/crispr.2022.0058"; journalName: "CRISPR J"; date: 
            "2022-10"; volume: "5"; issue: "5"; pages: "685-701"
COMMENT     Sequence Label: U6-FnCas12a-TLR-MCV1-gRNA
FEATURES             Location/Qualifiers
     source          1..3702
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     61..83
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     75..92
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     76..92
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     102..121
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        102..120
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     145..161
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     146..162
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        201..449
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     201..221
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     372..391
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(551..571)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(551..569)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(590..606)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(590..606)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(603..625)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    614..630
                     /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(638..644,645..662,663..668))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    683..704
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(821..838)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(992..1580)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1072..1091)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1800..1902
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1903..2562
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1969..1988)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3051..3155)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3181..3636)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3318..3339)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3530..3549
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
       61 cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg
      121 gcgaattggg taccgggccc cccctcgagg tcgacggtat cgataagctt gatatcgaat
      181 tcctgcagcc cgggggatcc gagggcctat ttcccatgat tccttcatat ttgcatatac
      241 gatacaaggc tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag
      301 tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat
      361 gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt
      421 tatatatctt gtggaaagga cgaaacaccg tctaagaact ttaaataatt tctactgttg
      481 tagatgcgag acaaatcacc tgcctcgttt ttttgcggcc gccaccgcgg tggagctcca
      541 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
      601 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
      661 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
      721 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
      781 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
      841 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
      901 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
      961 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     1021 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     1081 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     1141 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     1201 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     1261 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     1321 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     1381 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     1441 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     1501 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     1561 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     1621 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     1681 agatcctttt cgaccgaata aatacctgtg acggaagatc acttcgcaga ataaataaat
     1741 cctggtgtcc ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgagacgtt
     1801 gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact accgggcgta
     1861 ttttttgagt tgtcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact
     1921 ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag
     1981 tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag
     2041 accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg
     2101 atgaatgctc atccggaatt acgtatggca atgaaagacg gtgagctggt gatatgggat
     2161 agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg
     2221 agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt
     2281 tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca
     2341 gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc
     2401 ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg
     2461 ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat
     2521 gaattacaac agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat
     2581 tggtgccctt aaacgcctgg ttgctacgcc tgaataagtg ataataagcg gatgaatggc
     2641 agaaattcga aagcaaattc gacccggtcg tcggttcagg gcagggtcgt taaatagccg
     2701 cttatgtcta ttgctggttt accggtttat tgactaccgg aagcagtgtg accgtgtgct
     2761 tctcaaatgc ctgaggccag tttgctcagg ctctccccgt ggaggtaata attgacgata
     2821 tgatcctttt tttctgatca aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     2881 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     2941 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     3001 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     3061 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     3121 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     3181 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     3241 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     3301 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     3361 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
     3421 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
     3481 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
     3541 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
     3601 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
     3661 actgttggga agggcgatcg gtgcgggcct cttcgctatt ac
//
