LOCUS       sequence-308533-        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_1
FEATURES             Location/Qualifiers
     source          1..7625
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(15..20,21..38,39..45)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    53..69
                     /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     58..80
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     77..93
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     77..93
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     112..132
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        114..132
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        160..386
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             387..567
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    614..739
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1232..1465
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1650..1694
                     /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(2034..2282)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2092..2111)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2262..2282)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2324..2441
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2490..3000
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2919..2937
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3022..3621
                     /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(3022..3041)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3518..3538
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3637..4225
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3690..3710)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4108..4119)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4297..4530
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4608..4729
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4645..4664)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4699..4718
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4769..4904
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4924..4943)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4925..4943)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4953..4970)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4953..4969)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4962..4984)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5111..5566
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5198..5217)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5408..5429
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5592..5696
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5697..5765,5766..6557)
                     /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(5915..5934)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6728..7316
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7217..7236
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7470..7487
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7604..7625
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
       61 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
      121 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct
      181 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc
      241 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac
      301 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat
      361 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg
      421 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
      481 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
      541 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
      601 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
      661 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
      721 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
      781 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
      841 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
      901 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
      961 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     1021 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
     1081 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
     1141 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
     1201 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
     1261 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
     1321 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
     1381 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
     1441 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
     1501 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
     1561 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
     1621 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
     1681 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat
     1741 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag
     1801 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga
     1861 ggggacccag agaattcaaa aaaaagtgct tgaagggaac cagacatcta cacttagtag
     1921 aaattagcac cgactcggtg ccactttttc aagttgataa cggactagcc ttatttcaac
     1981 ttgctatgct gtttccagca tagctctgaa acgccgtgag gtgaggtgca gtcggtacct
     2041 cgtcctttcc acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc
     2101 atatgatagt ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac
     2161 tttctacgtc acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt
     2221 atctctctaa cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc
     2281 tcgaattcta gatcttgaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2341 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2401 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2461 gcagagatcc actttggcgc cggctcgagg gggcccgggt tgcgcctttt ccaaggcagc
     2521 cctgggtttg cgcagggacg cggctgctct gggcgtggtt ccgggaaacg cagcggcgcc
     2581 gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg tcacccggat cttcgccgct
     2641 acccttgtgg gccccccggc gacgcttcct gctccgcccc taagtcggga aggttccttg
     2701 cggttcgcgg cgtgccggac gtgacaaacg gaagccgcag ctcacactag taccctcgca
     2761 gacggacagc gccagggagc aatggcagcg cgccgaccgc gatgggctgt ggccaatagc
     2821 ggctgctcag cagggcgcgc cgagagcagc ggccgggaag gggcggtgcg ggaggcgggg
     2881 tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt gttccgcatt ctgcaagcct
     2941 ccggagcgca cgtcggcagt cggctccctc gttgaccgaa tcaccgacct ctctccccag
     3001 ggggatccac cggagcttac catgaccgag tacaagccca cggtgcgcct cgccacccgc
     3061 gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg
     3121 cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc
     3181 ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgccgtg
     3241 gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg
     3301 cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg
     3361 gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggtgt ttcgcccgac
     3421 caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc
     3481 gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg
     3541 ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg
     3601 acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt
     3661 tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct
     3721 gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg
     3781 tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc
     3841 gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt
     3901 cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc
     3961 gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg
     4021 ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg
     4081 cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc
     4141 ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg
     4201 atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg
     4261 tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac
     4321 gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct
     4381 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     4441 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     4501 ccttttagtc agtgtggaaa atctctagca gtacgtatag tagttcatgt catcttatta
     4561 ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg
     4621 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4681 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc
     4741 tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     4801 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     4861 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat
     4921 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     4981 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     5041 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     5101 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     5161 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     5221 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5281 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     5341 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5401 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     5461 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5521 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     5581 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5641 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5701 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5761 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5821 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5881 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5941 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     6001 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     6061 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6121 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6181 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6241 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6301 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6361 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6421 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6481 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6541 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6601 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6661 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6721 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6781 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6841 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6901 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6961 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     7021 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     7081 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     7141 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     7201 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     7261 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     7321 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     7381 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     7441 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     7501 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     7561 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     7621 ctcat
//
