LOCUS       sequence-308516-        7628 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 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_exon3_2_Lb
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1073..1306
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1491..1535
                     /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(1878..2126)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1936..1955)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2106..2126)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2168..2285
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2334..2844
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2763..2781
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2866..3465
                     /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(2866..2885)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3362..3382
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3481..4069
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3534..3554)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3952..3963)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4141..4374
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4452..4573
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4489..4508)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4543..4562
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4613..4748
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4768..4787)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4769..4787)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4797..4814)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4797..4813)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4806..4828)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4955..5410
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5042..5061)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5252..5273
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5436..5540
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5541..5609,5610..6401)
                     /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(5759..5778)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6572..7160
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7061..7080
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7314..7331
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7448..7469
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7484..7489,7490..7507,7508..7514)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7522..7538
                     /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     7527..7549
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7546..7562
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7546..7562
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7581..7601
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7583..7601
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca
      961 attggagaag tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac
     1021 ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt
     1081 tgttccttgg gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga
     1141 cggtacaggc cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg
     1201 ctattgaggc gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg
     1261 caagaatcct ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt
     1321 gctctggaaa actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat
     1381 ctctggaaca gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt
     1441 acacaagctt aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac
     1501 aagaattatt ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt
     1561 ggctgtggta tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag
     1621 tttttgctgt actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc
     1681 agacccacct cccaaccccg aggggaccca gagaattcaa aaaaaagcaa ttcctaaaag
     1741 gagtttatta tctacactta gtagaaatta gcaccgactc ggtgccactt tttcaagttg
     1801 ataacggact agccttattt caacttgcta tgctgtttcc agcatagctc tgaaacttgc
     1861 cctcatgttt cataagcggt acctcgtcct ttccacaaga tatataaagc caagaaatcg
     1921 aaatactttc aagttacggt aagcatatga tagtccattt taaaacataa ttttaaaact
     1981 gcaaactacc caagaaatta ttactttcta cgtcacgtat tttgtactaa tatctttgtg
     2041 tttacagtca aattaattct aattatctct ctaacagcct tgtatcgtat atgcaaatat
     2101 gaaggaatca tgggaaatag gccctcgaat tctagatctt gagacaaatg gcagtattca
     2161 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     2221 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     2281 attttcgggt ttattacagg gacagcagag atccactttg gcgccggctc gagggggccc
     2341 gggttgcgcc ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt
     2401 ggttccggga aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc
     2461 agcgtcaccc ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg
     2521 cccctaagtc gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc
     2581 gcagctcaca ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga
     2641 ccgcgatggg ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg
     2701 gaaggggcgg tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg
     2761 cggtgttccg cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac
     2821 cgaatcaccg acctctctcc ccagggggat ccaccggagc ttaccatgac cgagtacaag
     2881 cccacggtgc gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc
     2941 gcgttcgccg actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg
     3001 gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg
     3061 gtcgcggacg acggcgccgc cgtggcggtc tggaccacgc cggagagcgt cgaagcgggg
     3121 gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg
     3181 cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg
     3241 gccaccgtcg gtgtttcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc
     3301 cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc
     3361 cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc
     3421 gaaggaccgc gcacctggtg catgacccgc aagcccggtg cctgaacgcg ttaagtcgac
     3481 aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
     3541 ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
     3601 atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg
     3661 tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact
     3721 ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct
     3781 attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg
     3841 ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc
     3901 gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc
     3961 aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt
     4021 cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcg tcgactttaa
     4081 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     4141 tggaagggct aattcactcc caacgaagac aagatctgct ttttgcttgt actgggtctc
     4201 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
     4261 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
     4321 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtacgt
     4381 atagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa tgaatatcag
     4441 agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4501 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4561 tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc ccatcccgcc
     4621 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta
     4681 tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt
     4741 tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     4801 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     4861 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     4921 tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     4981 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     5041 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     5101 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     5161 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     5221 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5281 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5341 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5401 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5461 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5521 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5581 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5641 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5701 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5761 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5821 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5881 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5941 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6001 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6061 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6121 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6181 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6241 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6301 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6361 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6421 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6481 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     6541 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     6601 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     6661 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     6721 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     6781 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     6841 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     6901 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     6961 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7021 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7081 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7141 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7201 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     7261 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     7321 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
     7381 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
     7441 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
     7501 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
     7561 accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag ctggagctgc
     7621 aagcttaa
//
