LOCUS       sequence-300015-        7630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven hybrid guide (hg)RNA for
            deletion of HPRT1 exon 3 using SpCas9 and AsCas12a nucleases 
            (CHyMErA system).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7630)
  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 7630)
  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_exon3_2_As
FEATURES             Location/Qualifiers
     source          1..7630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        7..507
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     426..444
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             529..1128
                     /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(529..548)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1025..1045
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    1144..1732
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1197..1217)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1615..1626)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             1804..2037
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2115..2236
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2152..2171)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2206..2225
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2276..2411
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2431..2450)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2432..2450)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2460..2477)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2460..2476)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2469..2491)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2618..3073
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2705..2724)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2915..2936
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3099..3203
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3204..3272,3273..4064)
                     /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(3422..3441)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4235..4823
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4724..4743
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4977..4994
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5111..5132
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5147..5152,5153..5170,5171..5177)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5185..5201
                     /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     5190..5212
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5209..5225
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5209..5225
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5244..5264
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5246..5264
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5292..5518
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5519..5699
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5746..5871
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6364..6597
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6782..6826
                     /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(7168..7416)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(7226..7245)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(7396..7416)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    7461..7578
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
ORIGIN
        1 cccgggttgc gccttttcca aggcagccct gggtttgcgc agggacgcgg ctgctctggg
       61 cgtggttccg ggaaacgcag cggcgccgac cctgggtctc gcacattctt cacgtccgtt
      121 cgcagcgtca cccggatctt cgccgctacc cttgtgggcc ccccggcgac gcttcctgct
      181 ccgcccctaa gtcgggaagg ttccttgcgg ttcgcggcgt gccggacgtg acaaacggaa
      241 gccgcagctc acactagtac cctcgcagac ggacagcgcc agggagcaat ggcagcgcgc
      301 cgaccgcgat gggctgtggc caatagcggc tgctcagcag ggcgcgccga gagcagcggc
      361 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
      421 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
      481 gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat gaccgagtac
      541 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
      601 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
      661 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
      721 tgggtcgcgg acgacggcgc cgccgtggcg gtctggacca cgccggagag cgtcgaagcg
      781 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
      841 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
      901 ctggccaccg tcggtgtttc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
      961 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
     1021 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
     1081 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc
     1141 gacaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt
     1201 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc
     1261 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag
     1321 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc
     1381 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc
     1441 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg
     1501 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg
     1561 ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc
     1621 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt
     1681 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt
     1741 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     1801 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     1861 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     1921 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     1981 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     2041 cgtatagtag ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat
     2101 cagagagtga gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     2161 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     2221 tcatcaatgt atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc
     2281 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2341 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     2401 ttttggaggc ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca
     2461 ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc
     2521 cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc
     2581 ccttcccaac agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta
     2641 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     2701 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     2761 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     2821 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     2881 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     2941 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     3001 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     3061 acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     3121 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     3181 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3241 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3301 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3361 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3421 tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca
     3481 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3541 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3601 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3661 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3721 acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa
     3781 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3841 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3901 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3961 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     4021 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     4081 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     4141 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     4201 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4261 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4321 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4381 ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4441 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4501 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4561 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4621 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4681 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4741 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4801 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4861 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4921 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4981 agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt
     5041 ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc
     5101 gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc
     5161 ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct
     5221 atgaccatga ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc
     5281 tgcaagctta atgtagtctt atgcaatact cttgtagtct tgcaacatgg taacgatgag
     5341 ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag
     5401 gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga ttggacgaac
     5461 cactgaattg ccgcattgca gagatattgt atttaagtgc ctagctcgat acataaacgg
     5521 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     5581 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     5641 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     5701 tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg
     5761 actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca
     5821 aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag
     5881 cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa
     5941 tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct
     6001 ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt
     6061 cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg
     6121 catcaaagga tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa
     6181 aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga
     6241 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     6301 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     6361 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     6421 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     6481 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     6541 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     6601 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     6661 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     6721 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     6781 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     6841 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     6901 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     6961 attatcgttt cagacccacc tcccaacccc gaggggaccc agagaattca aaaaaaagca
     7021 attcctaaaa ggagtttatt atctacaaga gtagaaatta gcaccgactc ggtgccactt
     7081 tttcaagttg ataacggact agccttattt caacttgcta tgctgtttcc agcatagctc
     7141 tgaaacttgc cctcatgttt cataagcggt acctcgtcct ttccacaaga tatataaagc
     7201 caagaaatcg aaatactttc aagttacggt aagcatatga tagtccattt taaaacataa
     7261 ttttaaaact gcaaactacc caagaaatta ttactttcta cgtcacgtat tttgtactaa
     7321 tatctttgtg tttacagtca aattaattct aattatctct ctaacagcct tgtatcgtat
     7381 atgcaaatat gaaggaatca tgggaaatag gccctctctg aattctagat cttgagacaa
     7441 atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta cagtgcaggg
     7501 gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca aaaacaaatt
     7561 acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact ttggcgccgg
     7621 ctcgaggggg
//
