LOCUS       sequence-225058-        7856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LbCas12a-gRNA targeting TLR2.0.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7856)
  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 7856)
  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-LbCas12a-TLR-MCV1-gRNA
FEATURES             Location/Qualifiers
     source          1..7856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        285..533
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     285..305
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     456..475
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    625..742
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        791..1301
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1220..1238
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1323..1922
                     /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(1323..1342)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1819..1839
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2050..2283
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2355..2476
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2392..2411)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2446..2465
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2516..2651
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2671..2690)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2672..2690)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2700..2717)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2700..2716)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2709..2731)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2858..3313
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2945..2964)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3155..3176
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3339..3443
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3444..3512,3513..4304)
                     /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(3662..3681)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4475..5063
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4964..4983
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5217..5234
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5351..5372
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     mobile_element  5397..6164
                     /mobile_element_type="insertion sequence:IS1"
                     /label=IS1
                     /note="prokaryotic transposable element"
     promoter        join(6165..6169,6170..6187,6188..6194)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6202..6218
                     /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     6207..6229
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6226..6242
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6226..6242
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6261..6281
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6263..6281
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6309..6535
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6536..6716
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6763..6888
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7381..7614
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7799..7843
                     /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 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
       61 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
      121 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
      181 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac
      241 ggtatcgatc acgagactag cctcgagcgg ccgccccctt caccgagggc ctatttccca
      301 tgattccttc atatttgcat atacgataca aggctgttag agagataatt ggaattaatt
      361 tgactgtaaa cacaaagata ttagtacaaa atacgtgacg tagaaagtaa taatttcttg
      421 ggtagtttgc agttttaaaa ttatgtttta aaatggacta tcatatgctt accgtaactt
      481 gaaagtattt cgatttcttg gctttatata tcttgtggaa aggacgaaac acctaatttc
      541 tactaagtgt agatgcgaga caaatcacct gcctcgtttt tttgaattct cgacctcgag
      601 acaaatggca gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc
      661 aggggaaaga atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca
      721 aattacaaaa attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcc
      781 gcggctcgag ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac
      841 gcggctgctc tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat
      901 tcttcacgtc cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg
      961 cgacgcttcc tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga
     1021 cgtgacaaac ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag
     1081 caatggcagc gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg
     1141 ccgagagcag cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg
     1201 ccctgttcct gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag
     1261 tcggctccct cgttgaccga atcaccgacc tctctcccca gggggatcca ccggagctta
     1321 ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg
     1381 tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg
     1441 accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg
     1501 acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg
     1561 agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg
     1621 gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg
     1681 agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc aagggtctgg
     1741 gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc
     1801 tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg
     1861 ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct
     1921 gacgcccgcc ccacgacccg cagcgcccga ccgaaaggag cgcacgaccc catgcatcgg
     1981 tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     2041 aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta
     2101 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     2161 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     2221 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     2281 gcagtagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
     2341 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     2401 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     2461 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
     2521 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2581 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     2641 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     2701 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     2761 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     2821 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
     2881 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2941 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     3001 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     3061 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     3121 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     3181 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3241 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     3301 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3361 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3421 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3481 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3541 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3601 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3661 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3721 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3781 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3841 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3901 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3961 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     4021 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     4081 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     4141 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     4201 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4261 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4321 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4381 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4441 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4501 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4561 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4621 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4681 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4741 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4801 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4861 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4921 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4981 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     5041 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     5101 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     5161 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     5221 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     5281 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     5341 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttggta
     5401 atgactccaa cttattgata gtgttttatg ttcagataat gcccgatgac tttgtcatgc
     5461 agctccaccg attttgagaa cgacagcgac ttccgtccca gccgtgccag gtgctgcctc
     5521 agattcaggt tatgccgctc aattcgctgc gtatatcgct tgctgattac gtgcagcttt
     5581 cccttcaggc gggattcata cagcggccag ccatccgtca tccatatcac cacgtcaaag
     5641 ggtgacagca ggctcataag acgccccagc gtcgccatag tgcgttcacc gaatacgtgc
     5701 gcaacaaccg tcttccggag actgtcatac gcgtaaaaca gccagcgctg gcgcgattta
     5761 gccccgacat agccccactg ttcgtccatt tccgcgcaga cgatgacgtc actgcccggc
     5821 tgtatgcgcg aggttaccga ctgcggcctg agttttttaa gtgacgtaaa atcgtgttga
     5881 ggccaacgcc cataatgcgg gctgttgccc ggcatccaac gccattcatg gccatatcaa
     5941 tgattttctg gtgcgtaccg ggttgagaag cggtgtaagt gaactgcagt tgccatgttt
     6001 tacggcagtg agagcagaga tagcgctgat gtccggcggt gcttttgccg ttacgcacca
     6061 ccccgtcagt agctgaacag gagggacagc tgatagaaac agaagccact ggagcacctc
     6121 aaaaacacca tcatacacta aatcagtaag ttggcagcat caccttacac tttatgcttc
     6181 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     6241 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc
     6301 aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
     6361 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
     6421 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
     6481 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
     6541 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
     6601 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
     6661 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     6721 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     6781 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     6841 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     6901 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     6961 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     7021 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     7081 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     7141 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     7201 aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag gaggagatat
     7261 gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg
     7321 agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat
     7381 aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat
     7441 gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt
     7501 gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca
     7561 gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat
     7621 ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag
     7681 taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat
     7741 taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa
     7801 gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt ggttta
//
