LOCUS       sequence-225030-        7909 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  SpyCas9 sgRNA 2 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-sgRNA2
FEATURES             Location/Qualifiers
     source          1..7909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        178..426
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     178..198
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     349..368
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        447..522
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     misc_feature    571..688
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        737..1247
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1166..1184
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1269..1868
                     /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(1269..1288)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1765..1785
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1996..2229
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2301..2422
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2338..2357)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2392..2411
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2462..2597
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2617..2636)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2618..2636)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2646..2663)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2646..2662)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2655..2677)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2804..3259
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2891..2910)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3101..3122
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3285..3389
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3390..3458,3459..4250)
                     /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(3608..3627)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4421..5009
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4910..4929
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5163..5180
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5297..5318
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     mobile_element  5343..6110
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     promoter        join(6111..6115,6116..6133,6134..6140)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6148..6164
                     /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     6153..6175
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6172..6188
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6172..6188
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6207..6227
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6209..6227
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6255..6481
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6482..6662
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6709..6834
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7327..7560
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7745..7789
                     /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 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acccgacagg
       61 cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat tcgattagtg
      121 aacggatctc gacggtatcg atcacgagac tagcctcgag cggccgcccc cttcaccgag
      181 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata
      241 attggaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag
      301 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg
      361 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga
      421 aacaccgtta ccgtattcca cgaggcgtat tagagctaga aatagcaagt taatataagg
      481 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg aattctcgac
      541 ctcgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta
      601 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca
      661 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact
      721 ttggccgcgg ctcgaggggg ttggggttgc gccttttcca aggcagccct gggtttgcgc
      781 agggacgcgg ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggtctc
      841 gcacattctt cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc
      901 ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt
      961 gccggacgtg acaaacggaa gccgcacgtc tcactagtac cctcgcagac ggacagcgcc
     1021 agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag
     1081 ggcgcgccga gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag
     1141 tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt
     1201 cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg
     1261 agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca
     1321 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     1381 atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     1441 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     1501 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     1561 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     1621 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     1681 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     1741 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     1801 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     1861 gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg
     1921 catcggtacc tttaagacca atgacttaca aggcagctgt agatcttagc cactttttaa
     1981 aagaaaaggg gggactggaa gggctaattc actcccaacg aagacaagat ctgctttttg
     2041 cttgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     2101 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     2161 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     2221 tctctagcag tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
     2281 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
     2341 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     2401 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
     2461 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     2521 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     2581 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg
     2641 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     2701 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     2761 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     2821 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2881 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2941 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     3001 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     3061 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     3121 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     3181 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     3241 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     3301 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     3361 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     3421 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     3481 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     3541 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     3601 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     3661 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     3721 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     3781 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     3841 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     3901 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     3961 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     4021 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     4081 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     4141 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     4201 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     4261 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     4321 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     4381 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     4441 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     4501 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     4561 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     4621 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     4681 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     4741 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     4801 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     4861 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     4921 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     4981 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     5041 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     5101 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     5161 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     5221 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     5281 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     5341 ttggtaatga ctccaactta ttgatagtgt tttatgttca gataatgccc gatgactttg
     5401 tcatgcagct ccaccgattt tgagaacgac agcgacttcc gtcccagccg tgccaggtgc
     5461 tgcctcagat tcaggttatg ccgctcaatt cgctgcgtat atcgcttgct gattacgtgc
     5521 agctttccct tcaggcggga ttcatacagc ggccagccat ccgtcatcca tatcaccacg
     5581 tcaaagggtg acagcaggct cataagacgc cccagcgtcg ccatagtgcg ttcaccgaat
     5641 acgtgcgcaa caaccgtctt ccggagactg tcatacgcgt aaaacagcca gcgctggcgc
     5701 gatttagccc cgacatagcc ccactgttcg tccatttccg cgcagacgat gacgtcactg
     5761 cccggctgta tgcgcgaggt taccgactgc ggcctgagtt ttttaagtga cgtaaaatcg
     5821 tgttgaggcc aacgcccata atgcgggctg ttgcccggca tccaacgcca ttcatggcca
     5881 tatcaatgat tttctggtgc gtaccgggtt gagaagcggt gtaagtgaac tgcagttgcc
     5941 atgttttacg gcagtgagag cagagatagc gctgatgtcc ggcggtgctt ttgccgttac
     6001 gcaccacccc gtcagtagct gaacaggagg gacagctgat agaaacagaa gccactggag
     6061 cacctcaaaa acaccatcat acactaaatc agtaagttgg cagcatcacc ttacacttta
     6121 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     6181 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
     6241 agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat
     6301 gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt
     6361 aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg
     6421 aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa
     6481 cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     6541 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     6601 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     6661 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
     6721 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
     6781 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
     6841 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
     6901 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
     6961 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     7021 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     7081 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     7141 caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
     7201 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     7261 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     7321 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     7381 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     7441 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     7501 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     7561 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     7621 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     7681 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     7741 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     7801 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     7861 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttagg
//
