LOCUS       sequence-308587-        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_3
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(2..7,8..25,26..32)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    40..56
                     /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     45..67
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     64..80
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     64..80
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     99..119
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        101..119
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        147..373
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             374..554
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    601..726
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1219..1452
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1637..1681
                     /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(2024..2272)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2082..2101)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2252..2272)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2314..2431
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2480..2990
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2909..2927
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3012..3611
                     /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(3012..3031)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3508..3528
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3627..4215
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3680..3700)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4098..4109)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4287..4520
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4598..4719
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4635..4654)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4689..4708
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4759..4894
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4914..4933)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4915..4933)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4943..4960)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4943..4959)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4952..4974)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5101..5556
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5188..5207)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5398..5419
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5582..5686
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5687..5755,5756..6547)
                     /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(5905..5924)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6718..7306
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7207..7226
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7460..7477
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7594..7615
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
       61 acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg
      121 aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa
      181 catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg
      241 attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac
      301 atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc
      361 tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
      421 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
      481 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
      541 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
      601 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
      661 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
      721 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
      781 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
      841 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
      901 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
      961 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     1021 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag
     1081 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
     1141 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
     1201 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
     1261 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
     1321 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
     1381 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
     1441 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
     1501 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
     1561 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
     1621 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
     1681 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
     1741 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
     1801 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccagaga
     1861 attcaaaaaa aaattggtcc tgtgagtact tggtaatcta cacttagtag aaattagcac
     1921 cgactcggtg ccactttttc aagttgataa cggactagcc ttatttcaac ttgctatgct
     1981 gtttccagca tagctctgaa acttgttaga actgataggt gccggtacct cgtcctttcc
     2041 acaagatata taaagccaag aaatcgaaat actttcaagt tacggtaagc atatgatagt
     2101 ccattttaaa acataatttt aaaactgcaa actacccaag aaattattac tttctacgtc
     2161 acgtattttg tactaatatc tttgtgttta cagtcaaatt aattctaatt atctctctaa
     2221 cagccttgta tcgtatatgc aaatatgaag gaatcatggg aaataggccc tcgaattcta
     2281 gatcttgaga caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg
     2341 gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt
     2401 acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc
     2461 actttggcgc cggctcgagg gggcccgggt tgcgcctttt ccaaggcagc cctgggtttg
     2521 cgcagggacg cggctgctct gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt
     2581 ctcgcacatt cttcacgtcc gttcgcagcg tcacccggat cttcgccgct acccttgtgg
     2641 gccccccggc gacgcttcct gctccgcccc taagtcggga aggttccttg cggttcgcgg
     2701 cgtgccggac gtgacaaacg gaagccgcag ctcacactag taccctcgca gacggacagc
     2761 gccagggagc aatggcagcg cgccgaccgc gatgggctgt ggccaatagc ggctgctcag
     2821 cagggcgcgc cgagagcagc ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg
     2881 tagtgtgggc cctgttcctg cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca
     2941 cgtcggcagt cggctccctc gttgaccgaa tcaccgacct ctctccccag ggggatccac
     3001 cggagcttac catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc
     3061 ccagggccgt acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg
     3121 tcgatccgga ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg
     3181 tcgggctcga catcggcaag gtgtgggtcg cggacgacgg cgccgccgtg gcggtctgga
     3241 ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg
     3301 agttgagcgg ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc
     3361 ggcccaagga gcccgcgtgg ttcctggcca ccgtcggtgt ttcgcccgac caccagggca
     3421 agggtctggg cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc
     3481 ccgccttcct ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca
     3541 ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc
     3601 ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat
     3661 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     3721 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     3781 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     3841 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     3901 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     3961 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     4021 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     4081 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     4141 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     4201 gggccgcctc cccgcgtcga ctttaagacc aatgacttac aaggcagctg tagatcttag
     4261 ccacttttta aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga
     4321 tctgcttttt gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc
     4381 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     4441 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     4501 agtgtggaaa atctctagca gtacgtatag tagttcatgt catcttatta ttcagtattt
     4561 ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg cagcttataa
     4621 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     4681 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc tctagctatc
     4741 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4801 catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta
     4861 ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat tcgccctata
     4921 gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     4981 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     5041 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg
     5101 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5161 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5221 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5281 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     5341 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5401 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     5461 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5521 acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt
     5581 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5641 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5701 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5761 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5821 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5881 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5941 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     6001 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     6061 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     6121 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     6181 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     6241 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     6301 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6361 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     6421 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6481 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6541 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6601 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6661 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     6721 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     6781 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     6841 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     6901 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     6961 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     7021 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     7081 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     7141 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     7201 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     7261 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     7321 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt
     7381 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg
     7441 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
     7501 caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
     7561 cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc
     7621 accccagg
//
