LOCUS       sequence-225057-        3701 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 3701)
  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 3701)
  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..3701
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..457)
                     /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     351..370
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     584..606
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     598..615
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     599..615
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     625..644
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        625..643
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     668..684
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     669..685
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        724..972
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     724..744
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     895..914
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1074..1094)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1074..1092)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1113..1129)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1113..1129)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1126..1148)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1137..1153
                     /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(1161..1167,1168..1185,1186..1191))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1206..1227
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1344..1361)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1515..2103)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1595..1614)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2323..2425
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             2426..3085
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2492..2511)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(3573..3678)
                     /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 cactacgtga accatcaccc
      241 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
      301 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
      361 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
      421 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc
      481 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg
      541 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt
      601 aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt gggtaccggg
      661 ccccccctcg aggtcgacgg tatcgataag cttgatatcg aattcctgca gcccggggga
      721 tccgagggcc tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga
      781 gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt
      841 agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat
      901 catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa
      961 ggacgaaaca ccgtctaaga actttaaata atttctactg ttgtagatgc gagacaaatc
     1021 acctgcctcg tttttttgcg gccgccaccg cggtggagct ccagcttttg ttccctttag
     1081 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     1141 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     1201 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     1261 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     1321 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     1381 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     1441 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     1501 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     1561 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     1621 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     1681 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     1741 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     1801 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     1861 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     1921 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     1981 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     2041 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     2101 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     2161 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttcgaccga
     2221 ataaatacct gtgacggaag atcacttcgc agaataaata aatcctggtg tccctgttga
     2281 taccgggaag ccctgggcca acttttggcg aaaatgagac gttgatcggc acgtaagagg
     2341 ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg agttgtcgag
     2401 attttcagga gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga
     2461 tatatcccaa tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac
     2521 ctataaccag accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa
     2581 gcacaagttt tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga
     2641 attacgtatg gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta
     2701 caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga
     2761 tttccggcag tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc
     2821 ctatttccct aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag
     2881 tttcaccagt tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac
     2941 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
     3001 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg
     3061 cgatgagtgg cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacgcc
     3121 tggttgctac gcctgaataa gtgataataa gcggatgaat ggcagaaatt cgaaagcaaa
     3181 ttcgacccgg tcgtcggttc agggcagggt cgttaaatag ccgcttatgt ctattgctgg
     3241 tttacaagtt tattgactac cggaagcagt gtgaccgtgt gcttctcaaa tgcctgaggc
     3301 cagtttgctc aggctctccc cgtggaggta ataattgacg atatgatcct ttttttctga
     3361 tcaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     3421 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     3481 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     3541 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     3601 atttatcaag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     3661 acaaataggg gttccgcgca catttccccg aaaagtgcca c
//
