LOCUS       sequence-225059-        7856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AsCas12a-gRNA targeting TLR2.0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7856)
  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 7856)
  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: pLKO.1-puro-U6-AsCas12a-TLR-MCV1-gRNA
FEATURES             Location/Qualifiers
     source          1..7856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        181..429
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     181..201
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     352..371
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    521..638
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        687..1197
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1116..1134
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1219..1818
                     /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(1219..1238)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1715..1735
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1946..2179
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2251..2372
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2288..2307)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2342..2361
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2412..2547
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2567..2586)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2568..2586)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2596..2613)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2596..2612)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2605..2627)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2754..3209
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2841..2860)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3051..3072
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3235..3339
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3340..3408,3409..4200)
                     /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     complement(3558..3577)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4371..4959
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4860..4879
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5113..5130
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5247..5268
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     mobile_element  5293..6060
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     promoter        join(6061..6065,6066..6083,6084..6090)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6098..6114
                     /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)."
     primer_bind     6103..6125
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6122..6138
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6122..6138
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6157..6177
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6159..6177
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6205..6431
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6432..6612
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6659..6784
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7277..7510
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7695..7739
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
ORIGIN
        1 aggcagggat attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac
       61 aggcccgaag gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta
      121 gtgaacggat ctcgacggta tcgatcacga gactagcctc gagcggccgc ccccttcacc
      181 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
      241 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      301 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      361 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      421 cgaaacaccg taatttctac tcttgtagat gcgagacaaa tcacctgcct cgtttttttg
      481 aattctcgac ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga
      541 ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta
      601 aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca
      661 gagatccact ttggccgcgg ctcgaggggg ttggggttgc gccttttcca aggcagccct
      721 gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac
      781 cctgggtctc gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc
      841 cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg
      901 ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac
      961 ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc
     1021 tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt
     1081 ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg
     1141 gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagggg
     1201 gatccaccgg agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
     1261 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
     1321 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
     1381 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
     1441 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
     1501 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     1561 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     1621 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     1681 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     1741 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     1801 cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca
     1861 cgaccccatg catcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc
     1921 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat
     1981 ctgctttttg cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     2041 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     2101 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     2161 gtgtggaaaa tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt
     2221 gcaaagaaat gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac
     2281 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     2341 tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc
     2401 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     2461 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     2521 agtagtgagg aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc
     2581 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
     2641 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
     2701 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
     2761 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     2821 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     2881 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     2941 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3001 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3061 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3121 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3181 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     3241 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3301 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3361 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3421 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3481 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3541 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3601 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3661 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3721 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3781 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3841 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3901 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3961 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4021 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4081 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4141 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4201 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4261 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4321 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4381 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4441 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4501 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4561 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4621 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4681 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4741 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4801 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4861 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4921 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4981 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     5041 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5101 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     5161 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     5221 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
     5281 gctttacact ttggtaatga ctccaactta ttgatagtgt tttatgttca gataatgccc
     5341 gatgactttg tcatgcagct ccaccgattt tgagaacgac agcgacttcc gtcccagccg
     5401 tgccaggtgc tgcctcagat tcaggttatg ccgctcaatt cgctgcgtat atcgcttgct
     5461 gattacgtgc agctttccct tcaggcggga ttcatacagc ggccagccat ccgtcatcca
     5521 tatcaccacg tcaaagggtg acagcaggct cataagacgc cccagcgtcg ccatagtgcg
     5581 ttcaccgaat acgtgcgcaa caaccgtctt ccggagactg tcatacgcgt aaaacagcca
     5641 gcgctggcgc gatttagccc cgacatagcc ccactgttcg tccatttccg cgcagacgat
     5701 gacgtcactg cccggctgta tgcgcgaggt taccgactgc ggcctgagtt ttttaagtga
     5761 cgtaaaatcg tgttgaggcc aacgcccata atgcgggctg ttgcccggca tccaacgcca
     5821 ttcatggcca tatcaatgat tttctggtgc gtaccgggtt gagaagcggt gtaagtgaac
     5881 tgcagttgcc atgttttacg gcagtgagag cagagatagc gctgatgtcc ggcggtgctt
     5941 ttgccgttac gcaccacccc gtcagtagct gaacaggagg gacagctgat agaaacagaa
     6001 gccactggag cacctcaaaa acaccatcat acactaaatc agtaagttgg cagcatcacc
     6061 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     6121 acaggaaaca gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa
     6181 caaaagctgg agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca
     6241 tggtaacgat gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat
     6301 tggtggaagt aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat
     6361 ggattggacg aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc
     6421 gatacataaa cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac
     6481 tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg
     6541 cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga
     6601 aaatctctag cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct
     6661 ctctcgacgc aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact
     6721 ggtgagtacg ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag
     6781 cgtcagtatt aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg
     6841 gggaaagaaa aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt
     6901 cgcagttaat cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct
     6961 acaaccatcc cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac
     7021 cctctattgt gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat
     7081 agaggaagag caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac
     7141 ctggaggagg agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa
     7201 aaattgaacc attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa
     7261 aaagagcagt gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta
     7321 tgggcgcagc gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc
     7381 agcagcagaa caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag
     7441 tctggggcat caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc
     7501 aacagctcct ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt
     7561 ggaatgctag ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg
     7621 agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc
     7681 aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt
     7741 ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag
     7801 gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagtt
//
