LOCUS       sequence-308586-        7628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven hybrid guide (hg)RNA for
            deletion of MDM4 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_MDM4_exon_deletion_1
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    8..24
                     /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     13..35
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     32..48
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     32..48
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     67..87
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        69..87
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        115..341
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             342..522
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    569..694
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1187..1420
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1605..1649
                     /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(1992..2240)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2050..2069)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2220..2240)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2282..2399
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2448..2958
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2877..2895
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2980..3579
                     /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(2980..2999)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3476..3496
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3595..4183
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3648..3668)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4066..4077)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4255..4488
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4566..4687
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4603..4622)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4657..4676
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4727..4862
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4882..4901)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4883..4901)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4911..4928)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4911..4927)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4920..4942)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5069..5524
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5156..5175)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5366..5387
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5550..5654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5655..5723,5724..6515)
                     /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(5873..5892)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6686..7274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7175..7194
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7428..7445
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7562..7583
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7598..7603,7604..7621,7622..7628)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
ORIGIN
        1 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc
       61 aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg agctgcaagc ttaatgtagt
      121 cttatgcaat actcttgtag tcttgcaaca tggtaacgat gagttagcaa catgccttac
      181 aaggagagaa aaagcaccgt gcatgccgat tggtggaagt aaggtggtac gatcgtgcct
      241 tattaggaag gcaacagacg ggtctgacat ggattggacg aaccactgaa ttgccgcatt
      301 gcagagatat tgtatttaag tgcctagctc gatacataaa cgggtctctc tggttagacc
      361 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      421 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      481 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
      541 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
      601 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
      661 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
      721 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
      781 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
      841 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
      901 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
      961 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     1021 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
     1081 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
     1141 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
     1201 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
     1261 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
     1321 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
     1381 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
     1441 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
     1501 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     1561 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     1621 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     1681 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     1741 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     1801 acctcccaac cccgagggga cccagagaat tcaaaaaaaa tttctttgta gctattttag
     1861 tgtatctaca cttagtagaa attagcaccg actcggtgcc actttttcaa gttgataacg
     1921 gactagcctt atttcaactt gctatgctgt ttccagcata gctctgaaac ttattcagtg
     1981 gacatgtatt cggtacctcg tcctttccac aagatatata aagccaagaa atcgaaatac
     2041 tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa aactgcaaac
     2101 tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt tgtgtttaca
     2161 gtcaaattaa ttctaattat ctctctaaca gccttgtatc gtatatgcaa atatgaagga
     2221 atcatgggaa ataggccctc gaattctaga tcttgagaca aatggcagta ttcatccaca
     2281 attttaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata gtagacataa
     2341 tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt caaaattttc
     2401 gggtttatta cagggacagc agagatccac tttggcgccg gctcgagggg gcccgggttg
     2461 cgccttttcc aaggcagccc tgggtttgcg cagggacgcg gctgctctgg gcgtggttcc
     2521 gggaaacgca gcggcgccga ccctgggtct cgcacattct tcacgtccgt tcgcagcgtc
     2581 acccggatct tcgccgctac ccttgtgggc cccccggcga cgcttcctgc tccgccccta
     2641 agtcgggaag gttccttgcg gttcgcggcg tgccggacgt gacaaacgga agccgcagct
     2701 cacactagta ccctcgcaga cggacagcgc cagggagcaa tggcagcgcg ccgaccgcga
     2761 tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg agagcagcgg ccgggaaggg
     2821 gcggtgcggg aggcggggtg tggggcggta gtgtgggccc tgttcctgcc cgcgcggtgt
     2881 tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg gctccctcgt tgaccgaatc
     2941 accgacctct ctccccaggg ggatccaccg gagcttacca tgaccgagta caagcccacg
     3001 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc
     3061 gccgactacc ccgccacgcg ccacaccgtc gatccggacc gccacatcga gcgggtcacc
     3121 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg
     3181 gacgacggcg ccgccgtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg
     3241 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa
     3301 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc
     3361 gtcggtgttt cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga
     3421 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac
     3481 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgaggt gcccgaagga
     3541 ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa cgcgttaagt cgacaatcaa
     3601 cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt
     3661 acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct
     3721 ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc
     3781 gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg
     3841 ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc
     3901 acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc
     3961 actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt
     4021 gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca
     4081 gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt
     4141 cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcgtcgact ttaagaccaa
     4201 tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag
     4261 ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg tctctctggt
     4321 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
     4381 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
     4441 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt acgtatagta
     4501 gttcatgtca tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg
     4561 agaggaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4621 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4681 tatcttatca tgtctggctc tagctatccc gcccctaact ccgcccatcc cgcccctaac
     4741 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     4801 ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     4861 cctagggacg tacccaattc gccctatagt gagtcgtatt acgcgcgctc actggccgtc
     4921 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     4981 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     5041 cagttgcgca gcctgaatgg cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg
     5101 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     5161 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5221 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5281 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5341 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5401 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     5461 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     5521 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     5581 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     5641 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     5701 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     5761 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     5821 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     5881 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     5941 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     6001 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     6061 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     6121 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     6181 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     6241 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     6301 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     6361 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     6421 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     6481 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     6541 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     6601 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     6661 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     6721 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     6781 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     6841 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     6901 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     6961 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     7021 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     7081 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     7141 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     7201 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     7261 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     7321 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     7381 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     7441 gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     7501 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     7561 ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc
     7621 gtatgttg
//
