LOCUS       sequence-308578-        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_3
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     29..46
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    163..184
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(199..204,205..222,223..229)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    237..253
                     /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     242..264
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     261..277
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     261..277
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     296..316
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        298..316
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        344..570
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             571..751
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    798..923
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1416..1649
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1834..1878
                     /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(2221..2469)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2279..2298)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2449..2469)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2511..2628
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2677..3187
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3106..3124
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3209..3808
                     /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(3209..3228)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3705..3725
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3824..4412
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3877..3897)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4295..4306)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4484..4717
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4795..4916
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4832..4851)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4886..4905
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4956..5091
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5111..5130)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5112..5130)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5140..5157)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5140..5156)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5149..5171)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5298..5753
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5385..5404)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5595..5616
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5779..5883
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5884..5952,5953..6744)
                     /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(6102..6121)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6915..7503
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7404..7423
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
       61 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
      121 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtga gttagctcac
      181 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
      241 gagcggataa caatttcaca caggaaacag ctatgaccat gattacgcca agcgcgcaat
      301 taaccctcac taaagggaac aaaagctgga gctgcaagct taatgtagtc ttatgcaata
      361 ctcttgtagt cttgcaacat ggtaacgatg agttagcaac atgccttaca aggagagaaa
      421 aagcaccgtg catgccgatt ggtggaagta aggtggtacg atcgtgcctt attaggaagg
      481 caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg cagagatatt
      541 gtatttaagt gcctagctcg atacataaac gggtctctct ggttagacca gatctgagcc
      601 tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga
      661 gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga
      721 cccttttagt cagtgtggaa aatctctagc agtggcgccc gaacagggac ttgaaagcga
      781 aagggaaacc agaggagctc tctcgacgca ggactcggct tgctgaagcg cgcacggcaa
      841 gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt gactagcgga ggctagaagg
      901 agagagatgg gtgcgagagc gtcagtatta agcgggggag aattagatcg cgatgggaaa
      961 aaattcggtt aaggccaggg ggaaagaaaa aatataaatt aaaacatata gtatgggcaa
     1021 gcagggagct agaacgattc gcagttaatc ctggcctgtt agaaacatca gaaggctgta
     1081 gacaaatact gggacagcta caaccatccc ttcagacagg atcagaagaa cttagatcat
     1141 tatataatac agtagcaacc ctctattgtg tgcatcaaag gatagagata aaagacacca
     1201 aggaagcttt agacaagata gaggaagagc aaaacaaaag taagaccacc gcacagcaag
     1261 cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag aagtgaatta
     1321 tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa ggcaaagaga
     1381 agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct tgggttcttg
     1441 ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca ggccagacaa
     1501 ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga ggcgcaacag
     1561 catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat cctggctgtg
     1621 gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg aaaactcatt
     1681 tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga acagatttgg
     1741 aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag cttaatacac
     1801 tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt attggaatta
     1861 gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg gtatataaaa
     1921 ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc tgtactttct
     1981 atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca cctcccaacc
     2041 ccgaggggac ccagagaatt caaaaaaaat aaatgaacag cagtctgtag gaatctacac
     2101 ttagtagaaa ttagcaccga ctcggtgcca ctttttcaag ttgataacgg actagcctta
     2161 tttcaacttg ctatgctgtt tccagcatag ctctgaaact tattcagtgg acatgtattc
     2221 ggtacctcgt cctttccaca agatatataa agccaagaaa tcgaaatact ttcaagttac
     2281 ggtaagcata tgatagtcca ttttaaaaca taattttaaa actgcaaact acccaagaaa
     2341 ttattacttt ctacgtcacg tattttgtac taatatcttt gtgtttacag tcaaattaat
     2401 tctaattatc tctctaacag ccttgtatcg tatatgcaaa tatgaaggaa tcatgggaaa
     2461 taggccctcg aattctagat cttgagacaa atggcagtat tcatccacaa ttttaaaaga
     2521 aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat agcaacagac
     2581 atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttcg ggtttattac
     2641 agggacagca gagatccact ttggcgccgg ctcgaggggg cccgggttgc gccttttcca
     2701 aggcagccct gggtttgcgc agggacgcgg ctgctctggg cgtggttccg ggaaacgcag
     2761 cggcgccgac cctgggtctc gcacattctt cacgtccgtt cgcagcgtca cccggatctt
     2821 cgccgctacc cttgtgggcc ccccggcgac gcttcctgct ccgcccctaa gtcgggaagg
     2881 ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa gccgcagctc acactagtac
     2941 cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc cgaccgcgat gggctgtggc
     3001 caatagcggc tgctcagcag ggcgcgccga gagcagcggc cgggaagggg cggtgcggga
     3061 ggcggggtgt ggggcggtag tgtgggccct gttcctgccc gcgcggtgtt ccgcattctg
     3121 caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt gaccgaatca ccgacctctc
     3181 tccccagggg gatccaccgg agcttaccat gaccgagtac aagcccacgg tgcgcctcgc
     3241 cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc
     3301 cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga
     3361 actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc
     3421 cgccgtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat
     3481 cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg
     3541 cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggtgtttc
     3601 gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc
     3661 cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta
     3721 cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg
     3781 gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac ctctggatta
     3841 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     3901 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     3961 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     4021 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     4081 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     4141 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     4201 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     4261 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     4321 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     4381 gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat gacttacaag
     4441 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     4501 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
     4561 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     4621 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     4681 ctcagaccct tttagtcagt gtggaaaatc tctagcagta cgtatagtag ttcatgtcat
     4741 cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg
     4801 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     4861 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     4921 gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     4981 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     5041 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt
     5101 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     5161 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     5221 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     5281 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     5341 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     5401 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     5461 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     5521 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     5581 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     5641 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     5701 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     5761 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     5821 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     5881 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     5941 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     6001 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     6061 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     6121 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     6181 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6241 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6301 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     6361 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     6421 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     6481 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     6541 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     6601 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     6661 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     6721 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     6781 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     6841 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     6901 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     6961 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     7021 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     7081 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     7141 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     7201 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     7261 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     7321 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     7381 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     7441 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     7501 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     7561 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     7621 gctgatac
//
