LOCUS       sequence-308585-        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_2
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    109..234
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    727..960
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1145..1189
                     /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(1532..1780)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1590..1609)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1760..1780)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    1822..1939
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1988..2498
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2417..2435
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2520..3119
                     /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(2520..2539)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3016..3036
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3135..3723
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3188..3208)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3606..3617)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3795..4028
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4106..4227
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4143..4162)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4197..4216
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4267..4402
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4422..4441)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4423..4441)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4451..4468)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4451..4467)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4460..4482)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4609..5064
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4696..4715)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4906..4927
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5090..5194
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5195..5263,5264..6055)
                     /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(5413..5432)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6226..6814
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6715..6734
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6968..6985
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7102..7123
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7138..7143,7144..7161,7162..7168)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7176..7192
                     /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     7181..7203
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7200..7216
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7200..7216
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7235..7255
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7237..7255
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7283..7509
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             join(7510..7628,1..62)
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag
       61 cagtggcgcc cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc
      121 aggactcggc ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg
      181 ccaaaaattt tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt
      241 aagcggggga gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa
      301 aaatataaat taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat
      361 cctggcctgt tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc
      421 cttcagacag gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt
      481 gtgcatcaaa ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag
      541 caaaacaaaa gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg
      601 agatatgagg gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc
      661 attaggagta gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt
      721 gggaatagga gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc
      781 gtcaatgacg ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa
      841 caatttgctg agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat
      901 caagcagctc caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct
      961 ggggatttgg ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag
     1021 ttggagtaat aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag
     1081 agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca
     1141 agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt
     1201 taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt
     1261 aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc
     1321 accattatcg tttcagaccc acctcccaac cccgagggga cccagagaat tcaaaaaaaa
     1381 agcaactaca agtccttacc tttatctaca cttagtagaa attagcaccg actcggtgcc
     1441 actttttcaa gttgataacg gactagcctt atttcaactt gctatgctgt ttccagcata
     1501 gctctgaaac ctgattatag caacaagcta cggtacctcg tcctttccac aagatatata
     1561 aagccaagaa atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac
     1621 ataattttaa aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta
     1681 ctaatatctt tgtgtttaca gtcaaattaa ttctaattat ctctctaaca gccttgtatc
     1741 gtatatgcaa atatgaagga atcatgggaa ataggccctc gaattctaga tcttgagaca
     1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac tttggcgccg
     1981 gctcgagggg gcccgggttg cgccttttcc aaggcagccc tgggtttgcg cagggacgcg
     2041 gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct
     2101 tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga
     2161 cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt
     2221 gacaaacgga agccgcagct cacactagta ccctcgcaga cggacagcgc cagggagcaa
     2281 tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg
     2341 agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc
     2401 tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg
     2461 gctccctcgt tgaccgaatc accgacctct ctccccaggg ggatccaccg gagcttacca
     2521 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac
     2581 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gatccggacc
     2641 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     2701 tcggcaaggt gtgggtcgcg gacgacggcg ccgccgtggc ggtctggacc acgccggaga
     2761 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     2821 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     2881 ccgcgtggtt cctggccacc gtcggtgttt cgcccgacca ccagggcaag ggtctgggca
     2941 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     3001 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     3061 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgcctgaa
     3121 cgcgttaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
     3181 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
     3241 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
     3301 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
     3361 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
     3421 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
     3481 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
     3541 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
     3601 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
     3661 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
     3721 cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
     3781 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
     3841 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     3901 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     3961 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     4021 ctctagcagt acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa
     4081 gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata
     4141 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4201 tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact
     4261 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     4321 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     4381 tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt
     4441 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     4501 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     4561 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
     4621 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     4681 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     4741 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     4801 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     4861 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     4921 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     4981 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     5041 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5101 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5161 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5221 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5281 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5341 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5401 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5461 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5521 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5581 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5641 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5701 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     5761 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     5821 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     5881 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     5941 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6001 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6061 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6121 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6181 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6241 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6301 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6361 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6421 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6481 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6541 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6601 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6661 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6721 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     6781 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     6841 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     6901 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     6961 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     7021 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     7081 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     7141 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     7201 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
     7261 aaagctggag ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc ttgcaacatg
     7321 gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc atgccgattg
     7381 gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg tctgacatgg
     7441 attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg cctagctcga
     7501 tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     7561 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     7621 cgtctgtt
//
