LOCUS       sequence-308576-        7760 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven multiplexed hybrid guide
            (hg)RNA targeting TK1 using SpCas9 and three intergenic sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7760)
  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 7760)
  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_4
FEATURES             Location/Qualifiers
     source          1..7760
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     438..457
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     691..708
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    825..846
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(861..866,867..884,885..891)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    899..915
                     /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     904..926
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     923..939
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     923..939
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     958..978
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        960..978
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1006..1232
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1233..1413
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1460..1585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2078..2311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2496..2540
                     /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(3015..3263)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3073..3092)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3243..3263)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    3305..3422
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3471..3981
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3900..3918
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4003..4602
                     /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(4003..4022)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4499..4519
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4618..5206
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4671..4691)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5089..5100)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5278..5511
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5589..5710
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5626..5645)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5680..5699
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5750..5885
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5905..5924)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5906..5924)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5934..5951)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5934..5950)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5943..5965)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6092..6547
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6179..6198)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6389..6410
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6573..6677
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6678..6746,6747..7538)
                     /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(6896..6915)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7709..7760,1..537)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
       61 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
      121 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
      181 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
      241 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
      301 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
      361 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
      421 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
      481 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
      541 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
      601 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
      661 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
      721 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
      781 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
      841 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
      901 gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca
      961 attaaccctc actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa
     1021 tactcttgta gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga
     1081 aaaagcaccg tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa
     1141 ggcaacagac gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata
     1201 ttgtatttaa gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag
     1261 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     1321 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     1381 gaccctttta gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc
     1441 gaaagggaaa ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc
     1501 aagaggcgag gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa
     1561 ggagagagat gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga
     1621 aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc
     1681 aagcagggag ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg
     1741 tagacaaata ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc
     1801 attatataat acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac
     1861 caaggaagct ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca
     1921 agcggccgct gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat
     1981 tatataaata taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga
     2041 gaagagtggt gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct
     2101 tgggagcagc aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac
     2161 aattattgtc tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac
     2221 agcatctgtt gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg
     2281 tggaaagata cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca
     2341 tttgcaccac tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt
     2401 ggaatcacac gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac
     2461 actccttaat tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat
     2521 tagataaatg ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa
     2581 aattattcat aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt
     2641 ctatagtgaa tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa
     2701 ccccgagggg acccagagaa ttcaaaaaaa actctgtgtg ctcaaggggg gctaatctac
     2761 acttagtaga aattaggaga ggctggcaat attaaactat ctacacttag tagaaattag
     2821 aactggttat tgttcagtca ttatctacac ttagtagaaa ttattgtggg gttgtttgtt
     2881 aggctcatct acacttagta gaaattagca ccgactcggt gccacttttt caagttgata
     2941 acggactagc cttatttcaa cttgctatgc tgtttccagc atagctctga aactcgtcga
     3001 tgcctatgac agccggtacc tcgtcctttc cacaagatat ataaagccaa gaaatcgaaa
     3061 tactttcaag ttacggtaag catatgatag tccattttaa aacataattt taaaactgca
     3121 aactacccaa gaaattatta ctttctacgt cacgtatttt gtactaatat ctttgtgttt
     3181 acagtcaaat taattctaat tatctctcta acagccttgt atcgtatatg caaatatgaa
     3241 ggaatcatgg gaaataggcc ctcgaattct agatcttgag acaaatggca gtattcatcc
     3301 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     3361 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     3421 ttcgggttta ttacagggac agcagagatc cactttggcg ccggctcgag ggggcccggg
     3481 ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt
     3541 tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc
     3601 gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc
     3661 ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca
     3721 gctcacacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg
     3781 cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa
     3841 ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg
     3901 tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga
     3961 atcaccgacc tctctcccca gggggatcca ccggagctta ccatgaccga gtacaagccc
     4021 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     4081 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     4141 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     4201 gcggacgacg gcgccgccgt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     4261 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     4321 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     4381 accgtcggtg tttcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     4441 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     4501 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     4561 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaacgcgtta agtcgacaat
     4621 caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct
     4681 tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg
     4741 gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg
     4801 cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
     4861 tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt
     4921 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     4981 ggcactgaca attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc
     5041 tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat
     5101 ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
     5161 cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcgtcg actttaagac
     5221 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     5281 aagggctaat tcactcccaa cgaagacaag atctgctttt tgcttgtact gggtctctct
     5341 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     5401 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     5461 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtacgtata
     5521 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     5581 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5641 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5701 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct
     5761 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     5821 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     5881 aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     5941 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     6001 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     6061 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     6121 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     6181 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     6241 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     6301 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     6361 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     6421 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     6481 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     6541 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     6601 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     6661 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     6721 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     6781 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     6841 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     6901 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     6961 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     7021 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     7081 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     7141 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     7201 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     7261 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     7321 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     7381 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     7441 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     7501 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     7561 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     7621 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     7681 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     7741 gcttgcaaac aaaaaaacca
//
