LOCUS       sequence-308440-        7627 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 AsCas12a nucleases 
            (CHyMErA system).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7627)
  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 7627)
  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_2_As
FEATURES             Location/Qualifiers
     source          1..7627
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     107..124
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    241..262
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(277..282,283..300,301..307)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    315..331
                     /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     320..342
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     339..355
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     339..355
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     374..394
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        376..394
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        422..648
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             649..829
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    876..1001
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1494..1727
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1912..1956
                     /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(2298..2546)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2356..2375)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2526..2546)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2588..2705
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2754..3264
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3183..3201
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3286..3885
                     /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(3286..3305)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3782..3802
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3901..4489
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3954..3974)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4372..4383)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4561..4794
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4872..4993
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4909..4928)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4963..4982
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5033..5168
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5188..5207)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5189..5207)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5217..5234)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5217..5233)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5226..5248)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5375..5830
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5462..5481)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5672..5693
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5856..5960
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5961..6029,6030..6821)
                     /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(6179..6198)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6992..7580
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7481..7500
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
       61 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
      121 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
      181 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
      241 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
      301 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      361 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta
      421 atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat
      481 gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat
      541 cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg
      601 ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg
      661 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
      721 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
      781 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga
      841 acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg
      901 ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga
      961 ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa
     1021 ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa
     1081 aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag
     1141 aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat
     1201 cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga
     1261 tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta
     1321 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac
     1381 aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca
     1441 cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct
     1501 ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg
     1561 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg
     1621 gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag
     1681 gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt
     1741 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa
     1801 tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat
     1861 tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa
     1921 caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat
     1981 tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata
     2041 gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt
     2101 cagacccacc tcccaacccc gaggggaccc agagaattca aaaaaaagtg ggactctgta
     2161 gggaccagaa atctacaaga gtagaaatta gcaccgactc ggtgccactt tttcaagttg
     2221 ataacggact agccttattt caacttgcta tgctgtttcc agcatagctc tgaaactgta
     2281 actatatgtc ttagtccggt acctcgtcct ttccacaaga tatataaagc caagaaatcg
     2341 aaatactttc aagttacggt aagcatatga tagtccattt taaaacataa ttttaaaact
     2401 gcaaactacc caagaaatta ttactttcta cgtcacgtat tttgtactaa tatctttgtg
     2461 tttacagtca aattaattct aattatctct ctaacagcct tgtatcgtat atgcaaatat
     2521 gaaggaatca tgggaaatag gccctcgaat tctagatctt gagacaaatg gcagtattca
     2581 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     2641 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     2701 attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc gagggggccc
     2761 gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt
     2821 ggttccggga aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc
     2881 agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg
     2941 cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc
     3001 gcagctcaca ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga
     3061 ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg
     3121 gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg
     3181 cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac
     3241 cgaatcaccg acctctctcc ccagggggat ccaccggagc ttaccatgac cgagtacaag
     3301 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     3361 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     3421 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     3481 gtcgcggacg acggcgccgc cgtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     3541 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     3601 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     3661 gccaccgtcg gtgtttcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     3721 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     3781 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     3841 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac
     3901 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     3961 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     4021 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     4081 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     4141 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     4201 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     4261 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     4321 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     4381 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     4441 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa
     4501 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     4561 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     4621 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     4681 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     4741 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt
     4801 atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag
     4861 agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4921 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4981 tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc
     5041 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     5101 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     5161 tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     5221 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     5281 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     5341 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     5401 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     5461 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5521 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5581 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5641 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5701 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5761 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5821 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5881 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5941 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     6001 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     6061 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     6121 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6181 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6241 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6301 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6361 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6421 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6481 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6541 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6601 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6661 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6721 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6781 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6841 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6901 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6961 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     7021 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     7081 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     7141 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     7201 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7261 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7321 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7381 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7441 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7501 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7561 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7621 gctggcc
//
