LOCUS       sequence-221583-        7181 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 7181)
  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 7181)
  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..7181
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      41..629
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     530..549
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     783..800
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    917..938
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(953..958,959..976,977..983)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    991..1007
                     /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     996..1018
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1015..1031
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1015..1031
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1050..1070
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1052..1070
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1098..1324
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1325..1505
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1552..1677
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2170..2403
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2588..2632
                     /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"
     promoter        2930..3178
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2930..2950
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3101..3120
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3231..3323
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     misc_feature    3372..3489
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3538..4048
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3967..3985
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4070..4669
                     /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(4070..4089)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4566..4586
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4797..5030
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5102..5223
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5139..5158)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5193..5212
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5263..5398
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5418..5437)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5419..5437)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5447..5464)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5447..5463)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5456..5478)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5605..6060
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5692..5711)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5902..5923
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6086..6190
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6191..6259,6260..7051)
                     /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(6409..6428)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
       61 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
      121 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
      181 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
      241 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
      301 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
      361 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
      421 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
      481 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
      541 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
      601 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
      661 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
      721 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
      781 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
      841 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
      901 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
      961 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     1021 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
     1081 tggagctgca agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac
     1141 gatgagttag caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga
     1201 agtaaggtgg tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg
     1261 acgaaccact gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat
     1321 aaacgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     1381 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     1441 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     1501 tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga
     1561 cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt
     1621 acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt
     1681 attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag
     1741 aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt
     1801 aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca
     1861 tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc aaccctctat
     1921 tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa
     1981 gagcaaaaca aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg
     2041 aggagatatg agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga
     2101 accattagga gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc
     2161 agtgggaata ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc
     2221 agcgtcaatg acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca
     2281 gaacaatttg ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg
     2341 catcaagcag ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct
     2401 cctggggatt tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc
     2461 tagttggagt aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga
     2521 cagagaaatt aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca
     2581 gcaagaaaag aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg
     2641 gtttaacata acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt
     2701 ggtaggttta agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata
     2761 ttcaccatta tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg
     2821 aatagaagaa gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc
     2881 tcgacggtat cgatcacgag actagcctcg agcggccgcc cccttcaccg agggcctatt
     2941 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattggaat
     3001 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
     3061 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
     3121 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga
     3181 ggtttaccgt attccacgag gcgttgtagc tccctttctc atttcggaaa cgaaatgaga
     3241 accgttgcta caataaggcc gtctgaaaag atgtgccgca acgctctgcc ccttaaagct
     3301 tctgctttaa ggggcatcgt ttattttttt gaattctcga cctcgagaca aatggcagta
     3361 ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata
     3421 gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt
     3481 caaaattttc gggtttatta cagggacagc agagatccac tttggccgcg gctcgagggg
     3541 gttggggttg cgccttttcc aaggcagccc tgggtttgcg cagggacgcg gctgctctgg
     3601 gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct tcacgtccgt
     3661 tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga cgcttcctgc
     3721 tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt gacaaacgga
     3781 agccgcacgt ctcactagta ccctcgcaga cggacagcgc cagggagcaa tggcagcgcg
     3841 ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg agagcagcgg
     3901 ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc tgttcctgcc
     3961 cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg gctccctcgt
     4021 tgaccgaatc accgacctct ctccccaggg ggatccaccg gagcttacca tgaccgagta
     4081 caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc
     4141 cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga
     4201 gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt
     4261 gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc
     4321 gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc
     4381 cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt
     4441 cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt
     4501 gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc
     4561 gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt
     4621 gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgac gcccgcccca
     4681 cgacccgcag cgcccgaccg aaaggagcgc acgaccccat gcatcggtac ctttaagacc
     4741 aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga
     4801 agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg
     4861 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     4921 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg
     4981 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtagtagttc
     5041 atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag
     5101 gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     5161 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     5221 ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg
     5281 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     5341 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     5401 gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt
     5461 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     5521 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     5581 tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     5641 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     5701 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     5761 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     5821 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     5881 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     5941 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     6001 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     6061 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     6121 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     6181 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     6241 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     6301 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     6361 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     6421 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     6481 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     6541 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     6601 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     6661 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     6721 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     6781 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     6841 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     6901 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     6961 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     7021 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     7081 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     7141 taatctcatg accaaaatcc cttaacgtga gttttcgttc c
//
