LOCUS       sequence-308510-        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_2
FEATURES             Location/Qualifiers
     source          1..7625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    166..291
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    784..1017
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1202..1246
                     /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(1586..1834)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1644..1663)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1814..1834)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    1876..1993
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2042..2552
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2471..2489
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2574..3173
                     /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(2574..2593)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3070..3090
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3189..3777
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3242..3262)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3660..3671)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3849..4082
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4160..4281
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4197..4216)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4251..4270
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4321..4456
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4476..4495)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4477..4495)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4505..4522)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4505..4521)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4514..4536)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4663..5118
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4750..4769)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4960..4981
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5144..5248
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5249..5317,5318..6109)
                     /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(5467..5486)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6280..6868
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6769..6788
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7022..7039
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7156..7177
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7192..7197,7198..7215,7216..7222)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7230..7246
                     /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     7235..7257
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7254..7270
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7254..7270
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7289..7309
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7291..7309
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7337..7563
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             join(7564..7625,1..119)
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
       61 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
      121 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
      181 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
      241 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
      301 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
      361 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
      421 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
      481 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
      541 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
      601 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
      661 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
      721 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
      781 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
      841 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
      901 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
      961 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1021 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1081 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     1141 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     1201 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     1261 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     1321 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     1381 attatcgttt cagacccacc tcccaacccc gaggggaccc agagaattca aaaaaaacca
     1441 cttgcttacc aggccatatc tacacttagt agaaattagc accgactcgg tgccactttt
     1501 tcaagttgat aacggactag ccttatttca acttgctatg ctgtttccag catagctctg
     1561 aaacggcacg ggcaggactg ctgacggtac ctcgtccttt ccacaagata tataaagcca
     1621 agaaatcgaa atactttcaa gttacggtaa gcatatgata gtccatttta aaacataatt
     1681 ttaaaactgc aaactaccca agaaattatt actttctacg tcacgtattt tgtactaata
     1741 tctttgtgtt tacagtcaaa ttaattctaa ttatctctct aacagccttg tatcgtatat
     1801 gcaaatatga aggaatcatg ggaaataggc cctcgaattc tagatcttga gacaaatggc
     1861 agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag
     1921 aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa
     1981 aattcaaaat tttcgggttt attacaggga cagcagagat ccactttggc gccggctcga
     2041 gggggcccgg gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct
     2101 ctgggcgtgg ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt
     2161 ccgttcgcag cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc
     2221 ctgctccgcc cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa
     2281 cggaagccgc agctcacact agtaccctcg cagacggaca gcgccaggga gcaatggcag
     2341 cgcgccgacc gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca
     2401 gcggccggga aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc
     2461 tgcccgcgcg gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc
     2521 tcgttgaccg aatcaccgac ctctctcccc agggggatcc accggagctt accatgaccg
     2581 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc
     2641 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca
     2701 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     2761 aggtgtgggt cgcggacgac ggcgccgccg tggcggtctg gaccacgccg gagagcgtcg
     2821 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     2881 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     2941 ggttcctggc caccgtcggt gtttcgcccg accaccaggg caagggtctg ggcagcgccg
     3001 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     3061 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     3121 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt
     3181 aagtcgacaa tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact
     3241 atgttgctcc ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg
     3301 cttcccgtat ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg
     3361 aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa
     3421 cccccactgg ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc
     3481 ccctccctat tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg
     3541 ctcggctgtt gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt
     3601 ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt
     3661 cggccctcaa tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc
     3721 cgcgtcttcg ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc
     3781 gactttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa
     3841 ggggggactg gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac
     3901 tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     3961 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     4021 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     4081 cagtacgtat agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg
     4141 aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa
     4201 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4261 caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc
     4321 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4381 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     4441 ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc
     4501 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
     4561 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
     4621 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
     4681 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     4741 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4801 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4861 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4921 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     4981 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
     5041 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     5101 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     5161 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     5221 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     5281 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     5341 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     5401 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     5461 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     5521 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     5581 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     5641 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     5701 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     5761 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     5821 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     5881 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     5941 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     6001 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     6061 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     6121 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     6181 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     6241 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     6301 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     6361 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     6421 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6481 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6541 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6601 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6661 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6721 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6781 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6841 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6901 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6961 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     7021 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     7081 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     7141 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     7201 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     7261 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
     7321 ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa catggtaacg
     7381 atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg attggtggaa
     7441 gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac atggattgga
     7501 cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc tcgatacata
     7561 aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
     7621 ccact
//
