LOCUS       sequence-308520-        7627 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 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_exon3_1_As
FEATURES             Location/Qualifiers
     source          1..7627
                     /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(1650..1898)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1708..1727)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1878..1898)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    1940..2057
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2106..2616
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2535..2553
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2638..3237
                     /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(2638..2657)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3134..3154
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3253..3841
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3306..3326)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3724..3735)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3913..4146
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4224..4345
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4261..4280)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4315..4334
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4385..4520
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4540..4559)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4541..4559)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4569..4586)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4569..4585)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4578..4600)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4727..5182
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4814..4833)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5024..5045
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5208..5312
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5313..5381,5382..6173)
                     /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(5531..5550)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6344..6932
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6833..6852
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7086..7103
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7220..7241
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7256..7261,7262..7279,7280..7286)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7294..7310
                     /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     7299..7321
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7318..7334
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7318..7334
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7353..7373
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7355..7373
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7401..7627
                     /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 caaaaaaaag
     1501 taaagtacta gtgaatatgt taatctacaa gagtagaaat tagcaccgac tcggtgccac
     1561 tttttcaagt tgataacgga ctagccttat ttcaacttgc tatgctgttt ccagcatagc
     1621 tctgaaactt gccctcatgt ttcataagcg gtacctcgtc ctttccacaa gatatataaa
     1681 gccaagaaat cgaaatactt tcaagttacg gtaagcatat gatagtccat tttaaaacat
     1741 aattttaaaa ctgcaaacta cccaagaaat tattactttc tacgtcacgt attttgtact
     1801 aatatctttg tgtttacagt caaattaatt ctaattatct ctctaacagc cttgtatcgt
     1861 atatgcaaat atgaaggaat catgggaaat aggccctcga attctagatc ttgagacaaa
     1921 tggcagtatt catccacaat tttaaaagaa aaggggggat tggggggtac agtgcagggg
     1981 aaagaatagt agacataata gcaacagaca tacaaactaa agaattacaa aaacaaatta
     2041 caaaaattca aaattttcgg gtttattaca gggacagcag agatccactt tggcgccggc
     2101 tcgagggggc ccgggttgcg ccttttccaa ggcagccctg ggtttgcgca gggacgcggc
     2161 tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc ctgggtctcg cacattcttc
     2221 acgtccgttc gcagcgtcac ccggatcttc gccgctaccc ttgtgggccc cccggcgacg
     2281 cttcctgctc cgcccctaag tcgggaaggt tccttgcggt tcgcggcgtg ccggacgtga
     2341 caaacggaag ccgcagctca cactagtacc ctcgcagacg gacagcgcca gggagcaatg
     2401 gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct gctcagcagg gcgcgccgag
     2461 agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg gggcggtagt gtgggccctg
     2521 ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg agcgcacgtc ggcagtcggc
     2581 tccctcgttg accgaatcac cgacctctct ccccaggggg atccaccgga gcttaccatg
     2641 accgagtaca agcccacggt gcgcctcgcc acccgcgacg acgtccccag ggccgtacgc
     2701 accctcgccg ccgcgttcgc cgactacccc gccacgcgcc acaccgtcga tccggaccgc
     2761 cacatcgagc gggtcaccga gctgcaagaa ctcttcctca cgcgcgtcgg gctcgacatc
     2821 ggcaaggtgt gggtcgcgga cgacggcgcc gccgtggcgg tctggaccac gccggagagc
     2881 gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca tggccgagtt gagcggttcc
     2941 cggctggccg cgcagcaaca gatggaaggc ctcctggcgc cgcaccggcc caaggagccc
     3001 gcgtggttcc tggccaccgt cggtgtttcg cccgaccacc agggcaaggg tctgggcagc
     3061 gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg gggtgcccgc cttcctggag
     3121 acctccgcgc cccgcaacct ccccttctac gagcggctcg gcttcaccgt caccgccgac
     3181 gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc gcaagcccgg tgcctgaacg
     3241 cgttaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt
     3301 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct
     3361 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt
     3421 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac
     3481 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct
     3541 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca
     3601 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt
     3661 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc
     3721 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct
     3781 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg
     3841 cgtcgacttt aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag
     3901 aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt
     3961 gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     4021 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     4081 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     4141 ctagcagtac gtatagtagt tcatgtcatc ttattattca gtatttataa cttgcaaaga
     4201 aatgaatatc agagagtgag aggaacttgt ttattgcagc ttataatggt tacaaataaa
     4261 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4321 tgtccaaact catcaatgta tcttatcatg tctggctcta gctatcccgc ccctaactcc
     4381 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4441 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4501 aggaggcttt tttggaggcc tagggacgta cccaattcgc cctatagtga gtcgtattac
     4561 gcgcgctcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa
     4621 cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc
     4681 accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgggacgc gccctgtagc
     4741 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4801 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     4861 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     4921 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     4981 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5041 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5101 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     5161 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     5221 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     5281 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     5341 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     5401 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     5461 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     5521 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     5581 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     5641 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     5701 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     5761 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     5821 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     5881 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     5941 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     6001 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
     6061 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     6121 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     6181 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     6241 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     6301 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     6361 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     6421 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     6481 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     6541 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     6601 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     6661 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     6721 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     6781 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     6841 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     6901 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     6961 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     7021 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     7081 agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc
     7141 ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg
     7201 gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac
     7261 actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag
     7321 gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa
     7381 agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt
     7441 aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt
     7501 ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat
     7561 tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata
     7621 cataaac
//
