LOCUS       sequence-308437-        7628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven hybrid guide (hg)RNA for
            deletion of HPRT1 exon 2 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_HPRT1_exon2_1_Lb
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..181
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    228..353
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    846..1079
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1264..1308
                     /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(1651..1899)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1709..1728)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1879..1899)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    1941..2058
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2107..2617
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2536..2554
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2639..3238
                     /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(2639..2658)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3135..3155
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3254..3842
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3307..3327)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3725..3736)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3914..4147
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4225..4346
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4262..4281)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4316..4335
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4386..4521
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4541..4560)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4542..4560)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4570..4587)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4570..4586)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4579..4601)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4728..5183
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4815..4834)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5025..5046
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5209..5313
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5314..5382,5383..6174)
                     /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(5532..5551)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6345..6933
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6834..6853
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7087..7104
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7221..7242
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7257..7262,7263..7280,7281..7287)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7295..7311
                     /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     7300..7322
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7319..7335
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7319..7335
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7354..7374
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7356..7374
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7402..7628
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
ORIGIN
        1 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
       61 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
      121 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
      181 agtggcgccc gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca
      241 ggactcggct tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc
      301 caaaaatttt gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta
      361 agcgggggag aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa
      421 aatataaatt aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc
      481 ctggcctgtt agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc
      541 ttcagacagg atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg
      601 tgcatcaaag gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc
      661 aaaacaaaag taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga
      721 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
      781 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
      841 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
      901 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
      961 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1021 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1081 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1141 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     1201 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     1261 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     1321 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     1381 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     1441 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccagagaatt caaaaaaaaa
     1501 ggccctcctc ttttattttt aaatctacac ttagtagaaa ttagcaccga ctcggtgcca
     1561 ctttttcaag ttgataacgg actagcctta tttcaacttg ctatgctgtt tccagcatag
     1621 ctctgaaact gtaactatat gtcttagtcc ggtacctcgt cctttccaca agatatataa
     1681 agccaagaaa tcgaaatact ttcaagttac ggtaagcata tgatagtcca ttttaaaaca
     1741 taattttaaa actgcaaact acccaagaaa ttattacttt ctacgtcacg tattttgtac
     1801 taatatcttt gtgtttacag tcaaattaat tctaattatc tctctaacag ccttgtatcg
     1861 tatatgcaaa tatgaaggaa tcatgggaaa taggccctcg aattctagat cttgagacaa
     1921 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     1981 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     2041 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg
     2101 ctcgaggggg cccgggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg
     2161 ctgctctggg cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt
     2221 cacgtccgtt cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac
     2281 gcttcctgct ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg
     2341 acaaacggaa gccgcagctc acactagtac cctcgcagac ggacagcgcc agggagcaat
     2401 ggcagcgcgc cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga
     2461 gagcagcggc cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct
     2521 gttcctgccc gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg
     2581 ctccctcgtt gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat
     2641 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     2701 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     2761 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     2821 cggcaaggtg tgggtcgcgg acgacggcgc cgccgtggcg gtctggacca cgccggagag
     2881 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     2941 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     3001 cgcgtggttc ctggccaccg tcggtgtttc gcccgaccac cagggcaagg gtctgggcag
     3061 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     3121 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     3181 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac
     3241 gcgttaagtc gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct
     3301 taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc
     3361 tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct
     3421 ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga
     3481 cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc
     3541 tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac
     3601 aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt
     3661 tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt
     3721 cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc
     3781 tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc
     3841 gcgtcgactt taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa
     3901 gaaaaggggg gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct
     3961 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     4021 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     4081 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     4141 tctagcagta cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag
     4201 aaatgaatat cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa
     4261 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4321 ttgtccaaac tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc
     4381 cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa
     4441 ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt
     4501 gaggaggctt ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta
     4561 cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     4621 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     4681 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag
     4741 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4801 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4861 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4921 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     4981 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     5041 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     5101 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5161 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     5221 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     5281 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     5341 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     5401 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     5461 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     5521 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     5581 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     5641 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     5701 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     5761 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     5821 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     5881 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     5941 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     6001 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     6061 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     6121 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     6181 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     6241 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     6301 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     6361 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     6421 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     6481 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     6541 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     6601 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     6661 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     6721 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     6781 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     6841 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     6901 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     6961 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     7021 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     7081 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     7141 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     7201 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     7261 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     7321 ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa
     7381 aagctggagc tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg
     7441 taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg
     7501 tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga
     7561 ttggacgaac cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat
     7621 acataaac
//
