LOCUS       sequence-308583-        7848 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven multiplexed hybrid guide
            (hg)RNA targeting TK1 using SpCas9 and five intergenic sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7848)
  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 7848)
  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_6
FEATURES             Location/Qualifiers
     source          1..7848
                     /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(2098..2346)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2156..2175)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2326..2346)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2388..2505
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2554..3064
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2983..3001
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3086..3685
                     /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(3086..3105)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3582..3602
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3701..4289
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3754..3774)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4172..4183)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4361..4594
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4672..4793
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4709..4728)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4763..4782
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4833..4968
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4988..5007)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4989..5007)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5017..5034)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5017..5033)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5026..5048)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5175..5630
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5262..5281)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5472..5493
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5656..5760
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5761..5829,5830..6621)
                     /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(5979..5998)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6792..7380
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7281..7300
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7534..7551
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7668..7689
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7704..7709,7710..7727,7728..7734)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7742..7758
                     /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     7747..7769
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7766..7782
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7766..7782
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7801..7821
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7803..7821
                     /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 aaaaaactct gtgtgctcaa
     1741 ggggggctaa tctacactta gtagaaatta tctaggccgg gaccagtgct tttatctaca
     1801 cttagtagaa attacaataa agcatgacaa tcagtatatc tacacttagt agaaattagg
     1861 agaggctggc aatattaaac tatctacact tagtagaaat tagaactggt tattgttcag
     1921 tcattatcta cacttagtag aaattattgt ggggttgttt gttaggctca tctacactta
     1981 gtagaaatta gcaccgactc ggtgccactt tttcaagttg ataacggact agccttattt
     2041 caacttgcta tgctgtttcc agcatagctc tgaaactcgt cgatgcctat gacagccggt
     2101 acctcgtcct ttccacaaga tatataaagc caagaaatcg aaatactttc aagttacggt
     2161 aagcatatga tagtccattt taaaacataa ttttaaaact gcaaactacc caagaaatta
     2221 ttactttcta cgtcacgtat tttgtactaa tatctttgtg tttacagtca aattaattct
     2281 aattatctct ctaacagcct tgtatcgtat atgcaaatat gaaggaatca tgggaaatag
     2341 gccctcgaat tctagatctt gagacaaatg gcagtattca tccacaattt taaaagaaaa
     2401 ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc aacagacata
     2461 caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt ttattacagg
     2521 gacagcagag atccactttg gcgccggctc gagggggccc gggttgcgcc ttttccaagg
     2581 cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga aacgcagcgg
     2641 cgccgaccct gggtctcgca cattcttcac gtccgttcgc agcgtcaccc ggatcttcgc
     2701 cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc gggaaggttc
     2761 cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcagctcaca ctagtaccct
     2821 cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg ctgtggccaa
     2881 tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg tgcgggaggc
     2941 ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg cattctgcaa
     3001 gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg acctctctcc
     3061 ccagggggat ccaccggagc ttaccatgac cgagtacaag cccacggtgc gcctcgccac
     3121 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     3181 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     3241 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     3301 cgtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     3361 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     3421 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gtgtttcgcc
     3481 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     3541 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     3601 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     3661 catgacccgc aagcccggtg cctgaacgcg ttaagtcgac aatcaacctc tggattacaa
     3721 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     3781 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     3841 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     3901 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     3961 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     4021 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     4081 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     4141 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     4201 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     4261 tcggatctcc ctttgggccg cctccccgcg tcgactttaa gaccaatgac ttacaaggca
     4321 gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc
     4381 caacgaagac aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga
     4441 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     4501 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     4561 agaccctttt agtcagtgtg gaaaatctct agcagtacgt atagtagttc atgtcatctt
     4621 attattcagt atttataact tgcaaagaaa tgaatatcag agagtgagag gaacttgttt
     4681 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     4741 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     4801 tggctctagc tatcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     4861 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     4921 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta gggacgtacc
     4981 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg
     5041 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     5101 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     5161 gaatggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     5221 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     5281 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     5341 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     5401 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     5461 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     5521 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     5581 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     5641 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     5701 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     5761 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     5821 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     5881 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     5941 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     6001 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     6061 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     6121 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     6181 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     6241 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     6301 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     6361 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     6421 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     6481 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     6541 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     6601 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     6661 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     6721 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     6781 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     6841 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     6901 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     6961 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     7021 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     7081 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     7141 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     7201 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     7261 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     7321 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     7381 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     7441 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     7501 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     7561 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     7621 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     7681 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     7741 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagcgc
     7801 gcaattaacc ctcactaaag ggaacaaaag ctggagctgc aagcttaa
//
