LOCUS       sequence-225027-        7909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SpyCas9 sgRNA 1 targeting TLR2.0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7909)
  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 7909)
  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-SpyCas9-TLR-MCV1-sgRNA1
FEATURES             Location/Qualifiers
     source          1..7909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        200..448
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     200..220
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     371..390
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        469..544
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    593..710
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        759..1269
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1188..1206
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1291..1890
                     /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(1291..1310)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1787..1807
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2018..2251
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2323..2444
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2360..2379)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2414..2433
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2484..2619
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2639..2658)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2640..2658)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2668..2685)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2668..2684)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2677..2699)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2826..3281
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2913..2932)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3123..3144
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3307..3411
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3412..3480,3481..4272)
                     /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(3630..3649)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4443..5031
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4932..4951
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5185..5202
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5319..5340
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     mobile_element  5365..6132
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     promoter        join(6133..6137,6138..6155,6156..6162)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6170..6186
                     /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     6175..6197
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6194..6210
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6194..6210
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6229..6249
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6231..6249
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6277..6503
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6504..6684
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6731..6856
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7349..7582
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7767..7811
                     /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 ttctatagtg aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc
       61 aaccccgagg ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag
      121 agacagatcc attcgattag tgaacggatc tcgacggtat cgatcacgag actagcctcg
      181 agcggccgcc cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg
      241 atacaaggct gttagagaga taattggaat taatttgact gtaaacacaa agatattagt
      301 acaaaatacg tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg
      361 ttttaaaatg gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt
      421 atatatcttg tggaaaggac gaaacaccga gacaaatcac ctgcctcggt attagagcta
      481 gaaatagcaa gttaatataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
      541 gtgctttttt tgaattctcg acctcgagac aaatggcagt attcatccac aattttaaaa
      601 gaaaaggggg gattgggggg tacagtgcag gggaaagaat agtagacata atagcaacag
      661 acatacaaac taaagaatta caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt
      721 acagggacag cagagatcca ctttggccgc ggctcgaggg ggttggggtt gcgccttttc
      781 caaggcagcc ctgggtttgc gcagggacgc ggctgctctg ggcgtggttc cgggaaacgc
      841 agcggcgccg accctgggtc tcgcacattc ttcacgtccg ttcgcagcgt cacccggatc
      901 ttcgccgcta cccttgtggg ccccccggcg acgcttcctg ctccgcccct aagtcgggaa
      961 ggttccttgc ggttcgcggc gtgccggacg tgacaaacgg aagccgcacg tctcactagt
     1021 accctcgcag acggacagcg ccagggagca atggcagcgc gccgaccgcg atgggctgtg
     1081 gccaatagcg gctgctcagc agggcgcgcc gagagcagcg gccgggaagg ggcggtgcgg
     1141 gaggcggggt gtggggcggt agtgtgggcc ctgttcctgc ccgcgcggtg ttccgcattc
     1201 tgcaagcctc cggagcgcac gtcggcagtc ggctccctcg ttgaccgaat caccgacctc
     1261 tctccccagg gggatccacc ggagcttacc atgaccgagt acaagcccac ggtgcgcctc
     1321 gccacccgcg acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac
     1381 cccgccacgc gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa
     1441 gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc
     1501 gccgcggtgg cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag
     1561 atcggcccgc gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa
     1621 ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc
     1681 tcgcccgacc accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg
     1741 gccgagcgcg ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc
     1801 tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc
     1861 tggtgcatga cccgcaagcc cggtgcctga cgcccgcccc acgacccgca gcgcccgacc
     1921 gaaaggagcg cacgacccca tgcatcggta cctttaagac caatgactta caaggcagct
     1981 gtagatctta gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa
     2041 cgaagacaag atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc
     2101 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
     2161 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
     2221 cccttttagt cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag
     2281 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     2341 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     2401 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     2461 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     2521 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     2581 agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc
     2641 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     2701 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     2761 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     2821 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     2881 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     2941 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3001 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     3061 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     3121 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     3181 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     3241 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     3301 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     3361 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     3421 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     3481 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     3541 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     3601 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     3661 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     3721 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     3781 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     3841 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     3901 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     3961 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     4021 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     4081 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     4141 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     4201 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     4261 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     4321 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     4381 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     4441 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     4501 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     4561 ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac
     4621 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     4681 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     4741 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     4801 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     4861 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     4921 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     4981 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     5041 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     5101 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     5161 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
     5221 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     5281 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
     5341 taggcacccc aggctttaca ctttggtaat gactccaact tattgatagt gttttatgtt
     5401 cagataatgc ccgatgactt tgtcatgcag ctccaccgat tttgagaacg acagcgactt
     5461 ccgtcccagc cgtgccaggt gctgcctcag attcaggtta tgccgctcaa ttcgctgcgt
     5521 atatcgcttg ctgattacgt gcagctttcc cttcaggcgg gattcataca gcggccagcc
     5581 atccgtcatc catatcacca cgtcaaaggg tgacagcagg ctcataagac gccccagcgt
     5641 cgccatagtg cgttcaccga atacgtgcgc aacaaccgtc ttccggagac tgtcatacgc
     5701 gtaaaacagc cagcgctggc gcgatttagc cccgacatag ccccactgtt cgtccatttc
     5761 cgcgcagacg atgacgtcac tgcccggctg tatgcgcgag gttaccgact gcggcctgag
     5821 ttttttaagt gacgtaaaat cgtgttgagg ccaacgccca taatgcgggc tgttgcccgg
     5881 catccaacgc cattcatggc catatcaatg attttctggt gcgtaccggg ttgagaagcg
     5941 gtgtaagtga actgcagttg ccatgtttta cggcagtgag agcagagata gcgctgatgt
     6001 ccggcggtgc ttttgccgtt acgcaccacc ccgtcagtag ctgaacagga gggacagctg
     6061 atagaaacag aagccactgg agcacctcaa aaacaccatc atacactaaa tcagtaagtt
     6121 ggcagcatca ccttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     6181 taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct
     6241 cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt
     6301 agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc
     6361 gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga
     6421 cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta
     6481 agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc
     6541 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     6601 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     6661 agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa
     6721 accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga
     6781 ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga
     6841 tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg
     6901 gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga
     6961 gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat
     7021 actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa
     7081 tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc
     7141 tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc
     7201 tgatcttcag acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat
     7261 ataaagtagt aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg
     7321 tgcagagaga aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag
     7381 caggaagcac tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt
     7441 ctggtatagt gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt
     7501 tgcaactcac agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat
     7561 acctaaagga tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca
     7621 ctgctgtgcc ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca
     7681 cgacctggat ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa
     7741 ttgaagaatc gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat
     7801 gggcaagttt gtggaattgg tttaacataa caaattggct gtggtatata aaattattca
     7861 taatgatagt aggaggcttg gtaggtttaa gaatagtttt tgctgtact
//
