LOCUS       sequence-308577-        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_4
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      135..723
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     624..643
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     877..894
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1011..1032
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1047..1052,1053..1070,1071..1077)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1085..1101
                     /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     1090..1112
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1109..1125
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1109..1125
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1144..1164
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1146..1164
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1192..1418
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1419..1599
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1646..1771
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2264..2497
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2682..2726
                     /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(3069..3317)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3127..3146)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3297..3317)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    3359..3476
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3525..4035
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3954..3972
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4057..4656
                     /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(4057..4076)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4553..4573
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4672..5260
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4725..4745)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5143..5154)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5332..5565
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5643..5764
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5680..5699)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5734..5753
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5804..5939
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5959..5978)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5960..5978)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5988..6005)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5988..6004)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5997..6019)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6146..6601
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6233..6252)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6443..6464
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6627..6731
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6732..6800,6801..7592)
                     /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(6950..6969)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
       61 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
      121 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
      181 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
      241 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
      301 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
      361 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
      421 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
      481 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
      541 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
      601 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
      661 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
      721 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
      781 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
      841 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
      901 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
      961 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     1021 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     1081 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     1141 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt
     1201 atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag
     1261 gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat
     1321 taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca
     1381 gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga
     1441 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     1501 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     1561 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
     1621 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
     1681 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
     1741 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
     1801 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     1861 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     1921 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     1981 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     2041 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     2101 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     2161 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     2221 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     2281 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     2341 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     2401 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     2461 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     2521 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     2581 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     2641 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     2701 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     2761 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     2821 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
     2881 tcccaacccc gaggggaccc agagaattca aaaaaaatag ttctgacata ttctttccct
     2941 atctacactt agtagaaatt agcaccgact cggtgccact ttttcaagtt gataacggac
     3001 tagccttatt tcaacttgct atgctgtttc cagcatagct ctgaaactga ttatagcaac
     3061 aagctaccgg tacctcgtcc tttccacaag atatataaag ccaagaaatc gaaatacttt
     3121 caagttacgg taagcatatg atagtccatt ttaaaacata attttaaaac tgcaaactac
     3181 ccaagaaatt attactttct acgtcacgta ttttgtacta atatctttgt gtttacagtc
     3241 aaattaattc taattatctc tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc
     3301 atgggaaata ggccctcgaa ttctagatct tgagacaaat ggcagtattc atccacaatt
     3361 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     3421 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     3481 tttattacag ggacagcaga gatccacttt ggcgccggct cgagggggcc cgggttgcgc
     3541 cttttccaag gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg
     3601 aaacgcagcg gcgccgaccc tgggtctcgc acattcttca cgtccgttcg cagcgtcacc
     3661 cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt
     3721 cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcagctcac
     3781 actagtaccc tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg
     3841 gctgtggcca atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg
     3901 gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc
     3961 gcattctgca agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc
     4021 gacctctctc cccaggggga tccaccggag cttaccatga ccgagtacaa gcccacggtg
     4081 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     4141 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     4201 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     4261 gacggcgccg ccgtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     4321 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     4381 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     4441 ggtgtttcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     4501 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     4561 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     4621 cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga caatcaacct
     4681 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     4741 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     4801 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     4861 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     4921 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     4981 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     5041 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt
     5101 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     5161 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     5221 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga
     5281 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc
     5341 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag
     5401 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     5461 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     5521 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt
     5581 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga
     5641 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     5701 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     5761 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc
     5821 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     5881 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     5941 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt
     6001 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     6061 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     6121 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     6181 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     6241 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     6301 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     6361 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     6421 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     6481 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     6541 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     6601 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     6661 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     6721 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     6781 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     6841 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     6901 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     6961 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     7021 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     7081 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     7141 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     7201 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     7261 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     7321 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     7381 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     7441 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     7501 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     7561 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     7621 tttagatt
//
