LOCUS       sequence-308512-        7628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven hybrid guide (hg)RNA for
            deletion of SFRS7 exon using SpCas9 and LbCas12a nucleases (CHyMErA 
            system).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7628)
  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 7628)
  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_SFRS7_exon_deletion_4
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     119..136
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    253..274
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(289..294,295..312,313..319)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    327..343
                     /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     332..354
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     351..367
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     351..367
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     386..406
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        388..406
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        434..660
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             661..841
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    888..1013
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1506..1739
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1924..1968
                     /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(2311..2559)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2369..2388)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2539..2559)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2601..2718
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2767..3277
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3196..3214
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3299..3898
                     /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(3299..3318)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3795..3815
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3914..4502
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3967..3987)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4385..4396)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4574..4807
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4885..5006
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4922..4941)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4976..4995
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5046..5181
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5201..5220)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5202..5220)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5230..5247)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5230..5246)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5239..5261)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5388..5843
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5475..5494)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5685..5706
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5869..5973
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5974..6042,6043..6834)
                     /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(6192..6211)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7005..7593
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7494..7513
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
       61 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
      121 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
      181 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
      241 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
      301 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
      361 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctgga
      421 gctgcaagct taatgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg
      481 agttagcaac atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta
      541 aggtggtacg atcgtgcctt attaggaagg caacagacgg gtctgacatg gattggacga
      601 accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg atacataaac
      661 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
      721 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      781 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      841 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      901 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      961 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
     1021 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
     1081 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
     1141 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
     1201 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
     1261 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
     1321 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
     1381 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     1441 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     1501 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     1561 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     1621 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1681 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1741 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1801 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1861 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1921 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1981 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     2041 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     2101 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaatt cccaaaaaaa
     2161 aatgtcagac tgcacttaaa ctatctacac ttagtagaaa ttagcaccga ctcggtgcca
     2221 ctttttcaag ttgataacgg actagcctta tttcaacttg ctatgctgtt tccagcatag
     2281 ctctgaaacc actttgtatc tattgtacac ggtacctcgt cctttccaca agatatataa
     2341 agccaagaaa tcgaaatact ttcaagttac ggtaagcata tgatagtcca ttttaaaaca
     2401 taattttaaa actgcaaact acccaagaaa ttattacttt ctacgtcacg tattttgtac
     2461 taatatcttt gtgtttacag tcaaattaat tctaattatc tctctaacag ccttgtatcg
     2521 tatatgcaaa tatgaaggaa tcatgggaaa taggccctcg aattctagat cttgagacaa
     2581 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     2641 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     2701 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg
     2761 ctcgaggggg cccgggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg
     2821 ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt
     2881 cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac
     2941 gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg
     3001 acaaacggaa gccgcagctc acactagtac cctcgcagac ggacagcgcc agggagcaat
     3061 ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga
     3121 gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct
     3181 gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg
     3241 ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat
     3301 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     3361 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     3421 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     3481 cggcaaggtg tgggtcgcgg acgacggcgc cgccgtggcg gtctggacca cgccggagag
     3541 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     3601 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     3661 cgcgtggttc ctggccaccg tcggtgtttc gcccgaccac cagggcaagg gtctgggcag
     3721 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     3781 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     3841 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac
     3901 gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     3961 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     4021 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     4081 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     4141 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     4201 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     4261 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     4321 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     4381 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     4441 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     4501 gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     4561 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct
     4621 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     4681 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     4741 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     4801 tctagcagta cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     4861 aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa
     4921 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4981 ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc
     5041 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     5101 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     5161 gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta
     5221 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     5281 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     5341 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     5401 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     5461 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     5521 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     5581 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     5641 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5701 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     5761 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5821 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     5881 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5941 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     6001 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     6061 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     6121 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     6181 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     6241 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     6301 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     6361 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     6421 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     6481 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     6541 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     6601 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6661 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6721 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6781 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6841 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6901 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6961 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     7021 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     7081 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     7141 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     7201 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     7261 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     7321 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     7381 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     7441 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     7501 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     7561 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     7621 ttcctggc
//
