LOCUS       sequence-308442-        7628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven hybrid guide (hg)RNA 
            targeting TK1 using SpCas9 and HPRT1 using LbCas12a (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_TK1_HPRT1_Lb_1
FEATURES             Location/Qualifiers
     source          1..7628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    120..353
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             538..582
                     /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(925..1173)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(983..1002)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1153..1173)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    1215..1332
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1381..1891
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1810..1828
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1913..2512
                     /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(1913..1932)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2409..2429
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2528..3116
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2581..2601)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2999..3010)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3188..3421
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3499..3620
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3536..3555)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3590..3609
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3660..3795
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3815..3834)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3816..3834)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3844..3861)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3844..3860)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3853..3875)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4002..4457
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4089..4108)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4299..4320
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4483..4587
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4588..4656,4657..5448)
                     /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(4806..4825)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5619..6207
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6108..6127
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6361..6378
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6495..6516
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6531..6536,6537..6554,6555..6561)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6569..6585
                     /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     6574..6596
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6593..6609
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6593..6609
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6628..6648
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6630..6648
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6676..6902
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6903..7083
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    7130..7255
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
ORIGIN
        1 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
       61 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
      121 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
      181 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
      241 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
      301 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
      361 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
      421 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
      481 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
      541 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
      601 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
      661 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
      721 tcgtttcaga cccacctccc aaccccgagg ggacccagag aattcaaaaa aaactctgtg
      781 tgctcaaggg gggctaatct acacttagta gaaattagca ccgactcggt gccacttttt
      841 caagttgata acggactagc cttatttcaa cttgctatgc tgtttccagc atagctctga
      901 aactcgtcga tgcctatgac agccggtacc tcgtcctttc cacaagatat ataaagccaa
      961 gaaatcgaaa tactttcaag ttacggtaag catatgatag tccattttaa aacataattt
     1021 taaaactgca aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat
     1081 ctttgtgttt acagtcaaat taattctaat tatctctcta acagccttgt atcgtatatg
     1141 caaatatgaa ggaatcatgg gaaataggcc ctcgaattct agatcttgag acaaatggca
     1201 gtattcatcc acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga
     1261 atagtagaca taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa
     1321 attcaaaatt ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag
     1381 ggggcccggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc
     1441 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc
     1501 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc
     1561 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac
     1621 ggaagccgca gctcacacta gtaccctcgc agacggacag cgccagggag caatggcagc
     1681 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag
     1741 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct
     1801 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct
     1861 cgttgaccga atcaccgacc tctctcccca gggggatcca ccggagctta ccatgaccga
     1921 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     1981 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     2041 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     2101 ggtgtgggtc gcggacgacg gcgccgccgt ggcggtctgg accacgccgg agagcgtcga
     2161 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     2221 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     2281 gttcctggcc accgtcggtg tttcgcccga ccaccagggc aagggtctgg gcagcgccgt
     2341 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc
     2401 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     2461 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta
     2521 agtcgacaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     2581 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     2641 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     2701 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     2761 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     2821 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     2881 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg
     2941 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     3001 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     3061 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg
     3121 actttaagac caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag
     3181 gggggactgg aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact
     3241 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca
     3301 ctgcttaagc ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg
     3361 tgtgactctg gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc
     3421 agtacgtata gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga
     3481 atatcagaga gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3541 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3601 aaactcatca atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca
     3661 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3721 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     3781 gcttttttgg aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg
     3841 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     3901 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     3961 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc
     4021 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     4081 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     4141 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     4201 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     4261 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     4321 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     4381 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat
     4441 attaacgctt acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4501 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4561 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4621 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4681 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4741 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4801 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4861 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4921 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4981 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     5041 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     5101 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5161 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5221 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5281 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     5341 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5401 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5461 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5521 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5581 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5641 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5701 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5761 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5821 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5881 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5941 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6001 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6061 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6121 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6181 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6241 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     6301 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     6361 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     6421 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     6481 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     6541 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     6601 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
     6661 gagctgcaag cttaatgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga
     6721 tgagttagca acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag
     6781 taaggtggta cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac
     6841 gaaccactga attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacataa
     6901 acgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
     6961 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
     7021 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
     7081 gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc tctctcgacg
     7141 caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac
     7201 gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat
     7261 taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa
     7321 aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa
     7381 tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc
     7441 ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg
     7501 tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga
     7561 gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag
     7621 gagatatg
//
