LOCUS       sequence-225055-        7913 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CjeCas9 sgRNA targeting TLR2.0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7913)
  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 7913)
  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-CjeCas9-TLR-MCV1-sgRNA
FEATURES             Location/Qualifiers
     source          1..7913
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        333..581
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     333..353
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     504..523
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        605..677
                     /label=Cj gRNA scaffold
                     /note="guide RNA scaffold for the Campylobacter jejuni 
                     CRISPR/Cas9 system (Kim et al., 2016)"
     misc_feature    730..847
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        896..1406
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1325..1343
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1428..2027
                     /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(1428..1447)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1924..1944
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2155..2388
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2460..2581
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2497..2516)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2551..2570
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2621..2756
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2776..2795)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2777..2795)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2805..2822)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2805..2821)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2814..2836)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2963..3418
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3050..3069)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3260..3281
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3444..3548
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3549..3617,3618..4409)
                     /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(3767..3786)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4580..5168
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5069..5088
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5322..5339
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5456..5477
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     mobile_element  5502..6269
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     promoter        join(6270..6274,6275..6292,6293..6299)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6307..6323
                     /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     6312..6334
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6331..6347
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6331..6347
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6366..6386
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6368..6386
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6414..6640
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6641..6821
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6868..6993
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7486..7719
                     /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(7904..7913,1..35)
                     /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 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
       61 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
      121 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
      181 agacccacct cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg
      241 gagagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac
      301 gagactagcc tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat
      361 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
      421 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
      481 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
      541 atttcttggc tttatatatc ttgtggaaag gacgaaacac cggcgagaca aatcacctgc
      601 ctcggtttta gtccctgaaa agggactaaa ataaagagtt tgcgggactc tgcggggtta
      661 caatccccta aaaccgcttt ttttttttga attctcgacc tcgagacaaa tggcagtatt
      721 catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg aaagaatagt
      781 agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta caaaaattca
      841 aaattttcgg gtttattaca gggacagcag agatccactt tggccgcggc tcgagggggt
      901 tggggttgcg ccttttccaa ggcagccctg ggtttgcgca gggacgcggc tgctctgggc
      961 gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc acgtccgttc
     1021 gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc cccggcgacg cttcctgctc
     1081 cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga caaacggaag
     1141 ccgcacgtct cactagtacc ctcgcagacg gacagcgcca gggagcaatg gcagcgcgcc
     1201 gaccgcgatg ggctgtggcc aatagcggct gctcagcagg gcgcgccgag agcagcggcc
     1261 gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg ttcctgcccg
     1321 cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc tccctcgttg
     1381 accgaatcac cgacctctct ccccaggggg atccaccgga gcttaccatg accgagtaca
     1441 agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc accctcgccg
     1501 ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc cacatcgagc
     1561 gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt
     1621 gggtcgcgga cgacggcgcc gcggtggcgg tctggaccac gccggagagc gtcgaagcgg
     1681 gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc cggctggccg
     1741 cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc
     1801 tggccaccgt cggcgtctcg cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc
     1861 tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag acctccgcgc
     1921 cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc
     1981 ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgacgc ccgccccacg
     2041 acccgcagcg cccgaccgaa aggagcgcac gaccccatgc atcggtacct ttaagaccaa
     2101 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     2161 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt
     2221 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     2281 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     2341 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat
     2401 gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga
     2461 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2521 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2581 atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc
     2641 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     2701 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     2761 gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
     2821 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
     2881 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
     2941 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3001 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     3061 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3121 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     3181 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3241 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     3301 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     3361 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     3421 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     3481 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     3541 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     3601 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     3661 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     3721 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     3781 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     3841 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     3901 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     3961 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     4021 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     4081 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     4141 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     4201 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     4261 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     4321 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     4381 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     4441 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     4501 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     4561 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     4621 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     4681 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     4741 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     4801 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     4861 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     4921 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     4981 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     5041 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     5101 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     5161 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     5221 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     5281 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     5341 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     5401 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     5461 tgagttagct cactcattag gcaccccagg ctttacactt tggtaatgac tccaacttat
     5521 tgatagtgtt ttatgttcag ataatgcccg atgactttgt catgcagctc caccgatttt
     5581 gagaacgaca gcgacttccg tcccagccgt gccaggtgct gcctcagatt caggttatgc
     5641 cgctcaattc gctgcgtata tcgcttgctg attacgtgca gctttccctt caggcgggat
     5701 tcatacagcg gccagccatc cgtcatccat atcaccacgt caaagggtga cagcaggctc
     5761 ataagacgcc ccagcgtcgc catagtgcgt tcaccgaata cgtgcgcaac aaccgtcttc
     5821 cggagactgt catacgcgta aaacagccag cgctggcgcg atttagcccc gacatagccc
     5881 cactgttcgt ccatttccgc gcagacgatg acgtcactgc ccggctgtat gcgcgaggtt
     5941 accgactgcg gcctgagttt tttaagtgac gtaaaatcgt gttgaggcca acgcccataa
     6001 tgcgggctgt tgcccggcat ccaacgccat tcatggccat atcaatgatt ttctggtgcg
     6061 taccgggttg agaagcggtg taagtgaact gcagttgcca tgttttacgg cagtgagagc
     6121 agagatagcg ctgatgtccg gcggtgcttt tgccgttacg caccaccccg tcagtagctg
     6181 aacaggaggg acagctgata gaaacagaag ccactggagc acctcaaaaa caccatcata
     6241 cactaaatca gtaagttggc agcatcacct tacactttat gcttccggct cgtatgttgt
     6301 gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca
     6361 agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc
     6421 ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca
     6481 aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt
     6541 attaggaagg caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg
     6601 cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca
     6661 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     6721 cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag
     6781 atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac
     6841 ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg
     6901 cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga
     6961 ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg
     7021 cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata
     7081 gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca
     7141 gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa
     7201 cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata
     7261 aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc
     7321 gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag
     7381 aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa
     7441 ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct
     7501 tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca
     7561 ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga
     7621 ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat
     7681 cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg
     7741 aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga
     7801 acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag
     7861 cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aac
//
