LOCUS       sequence-225034-        7958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NmeCas9 sgRNA targeting TLR2.0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7958)
  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 7958)
  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-NmeCas9-TLR-MCV1-sgRNA
FEATURES             Location/Qualifiers
     source          1..7958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     misc_RNA        502..594
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     misc_feature    643..760
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        809..1319
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1238..1256
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1341..1940
                     /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(1341..1360)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1837..1857
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2068..2301
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2373..2494
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2410..2429)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2464..2483
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2534..2669
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2689..2708)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2690..2708)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2718..2735)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2718..2734)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2727..2749)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2876..3331
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2963..2982)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3173..3194
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3357..3461
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3462..3530,3531..4322)
                     /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(3680..3699)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4493..5081
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4982..5001
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5235..5252
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5369..5390
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     mobile_element  5415..6182
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     promoter        join(6183..6187,6188..6205,6206..6212)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6220..6236
                     /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     6225..6247
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6244..6260
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6244..6260
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6279..6299
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6281..6299
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6327..6553
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6554..6734
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6781..6906
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7399..7632
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7817..7861
                     /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 tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag acccacctcc
       61 caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga gagagagaca
      121 gagacagatc cattcgatta gtgaacggat ctcgacggta tcgatcacga gactagcctc
      181 gagcggccgc ccccttcacc 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 aggtttaccg tattccacga ggcgttgtag
      481 ctccctttct catttcggaa acgaaatgag aaccgttgct acaataaggc cgtctgaaaa
      541 gatgtgccgc aacgctctgc cccttaaagc ttctgcttta aggggcatcg tttatttttt
      601 tgaattctcg acctcgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg
      661 gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac
      721 taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag
      781 cagagatcca ctttggccgc ggctcgaggg ggttggggtt gcgccttttc caaggcagcc
      841 ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc agcggcgccg
      901 accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc ttcgccgcta
      961 cccttgtggg ccccccggcg acgcttcctg ctccgcccct aagtcgggaa ggttccttgc
     1021 ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg tctcactagt accctcgcag
     1081 acggacagcg ccagggagca atggcagcgc gccgaccgcg atgggctgtg gccaatagcg
     1141 gctgctcagc agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg gaggcggggt
     1201 gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc tgcaagcctc
     1261 cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat caccgacctc tctccccagg
     1321 gggatccacc ggagcttacc atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     1381 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     1441 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     1501 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     1561 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     1621 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     1681 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
     1741 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     1801 ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc
     1861 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     1921 cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg
     1981 cacgacccca tgcatcggta cctttaagac caatgactta caaggcagct gtagatctta
     2041 gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag
     2101 atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     2161 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     2221 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     2281 cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag tatttataac
     2341 ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt
     2401 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     2461 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc
     2521 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     2581 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     2641 gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag
     2701 tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     2761 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     2821 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg
     2881 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2941 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     3001 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     3061 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     3121 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     3181 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     3241 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     3301 aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg
     3361 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3421 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     3481 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     3541 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     3601 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     3661 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     3721 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     3781 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     3841 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     3901 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     3961 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     4021 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     4081 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     4141 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     4201 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     4261 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     4321 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     4381 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4441 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     4501 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     4561 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     4621 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     4681 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     4741 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     4801 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     4861 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     4921 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     4981 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5041 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5101 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     5161 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     5221 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     5281 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     5341 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     5401 aggctttaca ctttggtaat gactccaact tattgatagt gttttatgtt cagataatgc
     5461 ccgatgactt tgtcatgcag ctccaccgat tttgagaacg acagcgactt ccgtcccagc
     5521 cgtgccaggt gctgcctcag attcaggtta tgccgctcaa ttcgctgcgt atatcgcttg
     5581 ctgattacgt gcagctttcc cttcaggcgg gattcataca gcggccagcc atccgtcatc
     5641 catatcacca cgtcaaaggg tgacagcagg ctcataagac gccccagcgt cgccatagtg
     5701 cgttcaccga atacgtgcgc aacaaccgtc ttccggagac tgtcatacgc gtaaaacagc
     5761 cagcgctggc gcgatttagc cccgacatag ccccactgtt cgtccatttc cgcgcagacg
     5821 atgacgtcac tgcccggctg tatgcgcgag gttaccgact gcggcctgag ttttttaagt
     5881 gacgtaaaat cgtgttgagg ccaacgccca taatgcgggc tgttgcccgg catccaacgc
     5941 cattcatggc catatcaatg attttctggt gcgtaccggg ttgagaagcg gtgtaagtga
     6001 actgcagttg ccatgtttta cggcagtgag agcagagata gcgctgatgt ccggcggtgc
     6061 ttttgccgtt acgcaccacc ccgtcagtag ctgaacagga gggacagctg atagaaacag
     6121 aagccactgg agcacctcaa aaacaccatc atacactaaa tcagtaagtt ggcagcatca
     6181 ccttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     6241 acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg
     6301 aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa
     6361 catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg
     6421 attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac
     6481 atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc
     6541 tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     6601 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     6661 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     6721 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
     6781 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
     6841 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
     6901 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
     6961 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
     7021 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     7081 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     7141 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     7201 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag
     7261 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
     7321 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
     7381 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
     7441 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
     7501 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
     7561 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
     7621 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
     7681 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
     7741 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
     7801 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
     7861 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
     7921 aggaggcttg gtaggtttaa gaatagtttt tgctgtac
//
