LOCUS       sequence-308532-        7625 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven hybrid guide (hg)RNA for
            deletion of mouse Ptbp exon 8 using SpCas9 and LbCas12a nucleases 
            (CHyMErA system).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7625)
  AUTHORS   Gonatopoulos-Pournatzis T, Aregger M, Brown KR, Farhangmehr S, 
            Braunschweig U, Ward HN, Ha KCH, Weiss A, Billmann M, Durbic T, 
            Myers CL, Blencowe BJ, Moffat J
  TITLE     Genetic interaction mapping and exon-resolution functional genomics 
            with a hybrid Cas9-Cas12a platform.
  JOURNAL   Nat Biotechnol. 2020 May;38(5):638-648. doi: 
            10.1038/s41587-020-0437-z. Epub 2020 Mar 16.
  PUBMED    32249828
REFERENCE   2  (bases 1 to 7625)
  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: 
            "10.1038/s41587-020-0437-z"; journalName: "Nat Biotechnol"; date: 
            "2020-05"; volume: "38"; issue: "5"; pages: "638-648"
COMMENT     Sequence Label: pLCHKO_Ptbp_ex8_3
FEATURES             Location/Qualifiers
     source          1..7625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     336..355
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     589..606
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    723..744
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(759..764,765..782,783..789)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    797..813
                     /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     802..824
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     821..837
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     821..837
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     856..876
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        858..876
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        904..1130
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1131..1311
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1358..1483
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1976..2209
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2394..2438
                     /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"
     promoter        complement(2778..3026)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2836..2855)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3006..3026)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    3068..3185
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3234..3744
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3663..3681
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3766..4365
                     /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(3766..3785)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4262..4282
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4381..4969
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4434..4454)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4852..4863)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5041..5274
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5352..5473
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5389..5408)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5443..5462
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5513..5648
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5668..5687)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5669..5687)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5697..5714)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5697..5713)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5706..5728)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5855..6310
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5942..5961)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6152..6173
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6336..6440
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6441..6509,6510..7301)
                     /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(6659..6678)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7472..7625,1..435)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
       61 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
      121 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
      181 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
      241 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
      301 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
      361 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
      421 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
      481 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
      541 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
      601 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
      661 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
      721 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
      781 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
      841 gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga gctgcaagct
      901 taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac
      961 atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg
     1021 atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga accactgaat
     1081 tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac gggtctctct
     1141 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     1201 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     1261 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
     1321 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
     1381 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
     1441 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
     1501 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
     1561 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
     1621 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
     1681 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
     1741 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
     1801 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg
     1861 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1921 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1981 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     2041 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     2101 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     2161 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     2221 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     2281 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     2341 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     2401 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     2461 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     2521 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     2581 ttcagaccca cctcccaacc ccgaggggac ccagagaatt caaaaaaaag tctcagtaca
     2641 gcatcttaca tctacactta gtagaaatta gcaccgactc ggtgccactt tttcaagttg
     2701 ataacggact agccttattt caacttgcta tgctgtttcc agcatagctc tgaaacgtgg
     2761 catcccggtc tatggacggt acctcgtcct ttccacaaga tatataaagc caagaaatcg
     2821 aaatactttc aagttacggt aagcatatga tagtccattt taaaacataa ttttaaaact
     2881 gcaaactacc caagaaatta ttactttcta cgtcacgtat tttgtactaa tatctttgtg
     2941 tttacagtca aattaattct aattatctct ctaacagcct tgtatcgtat atgcaaatat
     3001 gaaggaatca tgggaaatag gccctcgaat tctagatctt gagacaaatg gcagtattca
     3061 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     3121 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     3181 attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc gagggggccc
     3241 gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt
     3301 ggttccggga aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc
     3361 agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg
     3421 cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc
     3481 gcagctcaca ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga
     3541 ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg
     3601 gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg
     3661 cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac
     3721 cgaatcaccg acctctctcc ccagggggat ccaccggagc ttaccatgac cgagtacaag
     3781 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     3841 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     3901 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     3961 gtcgcggacg acggcgccgc cgtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     4021 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     4081 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     4141 gccaccgtcg gtgtttcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     4201 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     4261 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     4321 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac
     4381 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     4441 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     4501 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     4561 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     4621 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     4681 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     4741 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     4801 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     4861 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     4921 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa
     4981 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     5041 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     5101 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     5161 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     5221 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt
     5281 atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag
     5341 agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     5401 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     5461 tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc
     5521 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     5581 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     5641 tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     5701 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     5761 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     5821 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     5881 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     5941 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     6001 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     6061 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     6121 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     6181 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     6241 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     6301 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     6361 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     6421 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     6481 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     6541 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     6601 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6661 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6721 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6781 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6841 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6901 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6961 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     7021 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     7081 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     7141 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     7201 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     7261 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     7321 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     7381 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     7441 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     7501 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     7561 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     7621 ttcta
//
