LOCUS       sequence-225056-        7911 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SauCas9 sgRNA targeting TLR2.0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7911)
  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 7911)
  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-SauCas9-TLR-MCV1-sgRNA
FEATURES             Location/Qualifiers
     source          1..7911
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        326..574
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     326..346
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     497..516
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        597..672
                     /label=Sa gRNA scaffold
                     /note="guide RNA scaffold for the Staphylococcus aureus 
                     CRISPR/Cas9 system"
     misc_feature    721..838
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        887..1397
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1316..1334
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1419..2018
                     /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(1419..1438)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1915..1935
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2146..2379
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2451..2572
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2488..2507)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2542..2561
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2612..2747
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2767..2786)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2768..2786)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2796..2813)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2796..2812)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2805..2827)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2954..3409
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3041..3060)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3251..3272
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3435..3539
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3540..3608,3609..4400)
                     /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(3758..3777)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4571..5159
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5060..5079
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5313..5330
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5447..5468
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     mobile_element  5493..6260
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     promoter        join(6261..6265,6266..6283,6284..6290)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6298..6314
                     /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     6303..6325
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6322..6338
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6322..6338
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6357..6377
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6359..6377
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6405..6631
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6632..6812
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6859..6984
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7477..7710
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             join(7895..7911,1..28)
                     /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 attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg
       61 gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc
      121 tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca
      181 cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag
      241 agacagagac agatccattc gattagtgaa cggatctcga cggtatcgat cacgagacta
      301 gcctcgagcg gccgccccct tcaccgaggg cctatttccc atgattcctt catatttgca
      361 tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa acacaaagat
      421 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
      481 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
      541 ggctttatat atcttgtgga aaggacgaaa caccgcgaga caaatcacct gcctcggttt
      601 tagtactctg gaaacagaat ctactaaaac aaggcaaaat gccgtgttta tctcgtcaac
      661 ttgttggcga gatttttttg aattctcgac ctcgagacaa atggcagtat tcatccacaa
      721 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
      781 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg
      841 ggtttattac agggacagca gagatccact ttggccgcgg ctcgaggggg ttggggttgc
      901 gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg cgtggttccg
      961 ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt cgcagcgtca
     1021 cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa
     1081 gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa gccgcacgtc
     1141 tcactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat
     1201 gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc cgggaagggg
     1261 cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt
     1321 ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca
     1381 ccgacctctc tccccagggg gatccaccgg agcttaccat gaccgagtac aagcccacgg
     1441 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     1501 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     1561 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     1621 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     1681 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     1741 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     1801 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     1861 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     1921 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     1981 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac gacccgcagc
     2041 gcccgaccga aaggagcgca cgaccccatg catcggtacc tttaagacca atgacttaca
     2101 aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa gggctaattc
     2161 actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg ttagaccaga
     2221 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     2281 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     2341 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca tgtcatctta
     2401 ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg aacttgttta
     2461 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     2521 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     2581 ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     2641 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     2701 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag ggacgtaccc
     2761 aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt
     2821 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
     2881 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
     2941 aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3001 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3061 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3121 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     3181 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3241 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
     3301 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     3361 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt
     3421 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     3481 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     3541 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3601 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     3661 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     3721 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     3781 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     3841 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     3901 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     3961 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     4021 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     4081 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     4141 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     4201 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     4261 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     4321 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     4381 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     4441 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     4501 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4561 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4621 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4681 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     4741 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4801 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4861 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4921 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4981 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     5041 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     5101 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     5161 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     5221 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     5281 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     5341 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     5401 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     5461 tcactcatta ggcaccccag gctttacact ttggtaatga ctccaactta ttgatagtgt
     5521 tttatgttca gataatgccc gatgactttg tcatgcagct ccaccgattt tgagaacgac
     5581 agcgacttcc gtcccagccg tgccaggtgc tgcctcagat tcaggttatg ccgctcaatt
     5641 cgctgcgtat atcgcttgct gattacgtgc agctttccct tcaggcggga ttcatacagc
     5701 ggccagccat ccgtcatcca tatcaccacg tcaaagggtg acagcaggct cataagacgc
     5761 cccagcgtcg ccatagtgcg ttcaccgaat acgtgcgcaa caaccgtctt ccggagactg
     5821 tcatacgcgt aaaacagcca gcgctggcgc gatttagccc cgacatagcc ccactgttcg
     5881 tccatttccg cgcagacgat gacgtcactg cccggctgta tgcgcgaggt taccgactgc
     5941 ggcctgagtt ttttaagtga cgtaaaatcg tgttgaggcc aacgcccata atgcgggctg
     6001 ttgcccggca tccaacgcca ttcatggcca tatcaatgat tttctggtgc gtaccgggtt
     6061 gagaagcggt gtaagtgaac tgcagttgcc atgttttacg gcagtgagag cagagatagc
     6121 gctgatgtcc ggcggtgctt ttgccgttac gcaccacccc gtcagtagct gaacaggagg
     6181 gacagctgat agaaacagaa gccactggag cacctcaaaa acaccatcat acactaaatc
     6241 agtaagttgg cagcatcacc ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     6301 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa
     6361 ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt cttatgcaat
     6421 actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac aaggagagaa
     6481 aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct tattaggaag
     6541 gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt gcagagatat
     6601 tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc agatctgagc
     6661 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     6721 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     6781 acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cttgaaagcg
     6841 aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc gcgcacggca
     6901 agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg aggctagaag
     6961 gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc gcgatgggaa
     7021 aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat agtatgggca
     7081 agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc agaaggctgt
     7141 agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga acttagatca
     7201 ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat aaaagacacc
     7261 aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac cgcacagcaa
     7321 gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga gaagtgaatt
     7381 atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca aggcaaagag
     7441 aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc ttgggttctt
     7501 gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac aggccagaca
     7561 attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg aggcgcaaca
     7621 gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa tcctggctgt
     7681 ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg gaaaactcat
     7741 ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg aacagatttg
     7801 gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa gcttaataca
     7861 ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat t
//
