LOCUS       sequence-308527-        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_4
FEATURES             Location/Qualifiers
     source          1..7625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      38..626
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     527..546
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     780..797
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    914..935
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(950..955,956..973,974..980)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    988..1004
                     /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     993..1015
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1012..1028
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1012..1028
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1047..1067
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1049..1067
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1095..1321
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1322..1502
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1549..1674
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2167..2400
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2585..2629
                     /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(2969..3217)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3027..3046)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3197..3217)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    3259..3376
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3425..3935
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3854..3872
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3957..4556
                     /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(3957..3976)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4453..4473
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4572..5160
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4625..4645)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5043..5054)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5232..5465
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5543..5664
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5580..5599)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5634..5653
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5704..5839
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5859..5878)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5860..5878)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5888..5905)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5888..5904)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5897..5919)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6046..6501
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6133..6152)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6343..6364
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6527..6631
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6632..6700,6701..7492)
                     /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(6850..6869)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
       61 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
      121 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
      181 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
      241 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
      301 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
      361 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
      421 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
      481 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
      541 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
      601 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
      661 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
      721 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
      781 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
      841 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
      901 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
      961 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     1021 gctatgacca tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg
     1081 agctgcaagc ttaatgtagt cttatgcaat actcttgtag tcttgcaaca tggtaacgat
     1141 gagttagcaa catgccttac aaggagagaa aaagcaccgt gcatgccgat tggtggaagt
     1201 aaggtggtac gatcgtgcct tattaggaag gcaacagacg ggtctgacat ggattggacg
     1261 aaccactgaa ttgccgcatt gcagagatat tgtatttaag tgcctagctc gatacataaa
     1321 cgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc
     1381 actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt
     1441 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
     1501 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
     1561 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
     1621 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
     1681 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
     1741 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
     1801 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
     1861 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
     1921 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
     1981 caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
     2041 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
     2101 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
     2161 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
     2221 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
     2281 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
     2341 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
     2401 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     2461 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     2521 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     2581 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     2641 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     2701 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     2761 accattatcg tttcagaccc acctcccaac cccgagggga cccagagaat tcaaaaaaaa
     2821 atgccgggcg cacctagaaa atctacactt agtagaaatt agcaccgact cggtgccact
     2881 ttttcaagtt gataacggac tagccttatt tcaacttgct atgctgtttc cagcatagct
     2941 ctgaaacctg gcgaggacga ggacgcacgg tacctcgtcc tttccacaag atatataaag
     3001 ccaagaaatc gaaatacttt caagttacgg taagcatatg atagtccatt ttaaaacata
     3061 attttaaaac tgcaaactac ccaagaaatt attactttct acgtcacgta ttttgtacta
     3121 atatctttgt gtttacagtc aaattaattc taattatctc tctaacagcc ttgtatcgta
     3181 tatgcaaata tgaaggaatc atgggaaata ggccctcgaa ttctagatct tgagacaaat
     3241 ggcagtattc atccacaatt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga
     3301 aagaatagta gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac
     3361 aaaaattcaa aattttcggg tttattacag ggacagcaga gatccacttt ggcgccggct
     3421 cgagggggcc cgggttgcgc cttttccaag gcagccctgg gtttgcgcag ggacgcggct
     3481 gctctgggcg tggttccggg aaacgcagcg gcgccgaccc tgggtctcgc acattcttca
     3541 cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc
     3601 ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac
     3661 aaacggaagc cgcagctcac actagtaccc tcgcagacgg acagcgccag ggagcaatgg
     3721 cagcgcgccg accgcgatgg gctgtggcca atagcggctg ctcagcaggg cgcgccgaga
     3781 gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt
     3841 tcctgcccgc gcggtgttcc gcattctgca agcctccgga gcgcacgtcg gcagtcggct
     3901 ccctcgttga ccgaatcacc gacctctctc cccaggggga tccaccggag cttaccatga
     3961 ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca
     4021 ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc
     4081 acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg
     4141 gcaaggtgtg ggtcgcggac gacggcgccg ccgtggcggt ctggaccacg ccggagagcg
     4201 tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc
     4261 ggctggccgc gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg
     4321 cgtggttcct ggccaccgtc ggtgtttcgc ccgaccacca gggcaagggt ctgggcagcg
     4381 ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga
     4441 cctccgcgcc ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg
     4501 tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc
     4561 gttaagtcga caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     4621 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     4681 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     4741 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     4801 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     4861 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     4921 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     4981 cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     5041 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     5101 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     5161 gtcgacttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     5221 aaagggggga ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg
     5281 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa
     5341 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct
     5401 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc
     5461 tagcagtacg tatagtagtt catgtcatct tattattcag tatttataac ttgcaaagaa
     5521 atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt acaaataaag
     5581 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     5641 gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc cctaactccg
     5701 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     5761 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     5821 ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag tcgtattacg
     5881 cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac
     5941 ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca
     6001 ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg
     6061 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     6121 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     6181 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     6241 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
     6301 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     6361 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
     6421 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     6481 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
     6541 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     6601 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     6661 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     6721 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     6781 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     6841 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     6901 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     6961 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     7021 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     7081 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     7141 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     7201 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     7261 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     7321 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     7381 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     7441 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     7501 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     7561 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     7621 ccact
//
