LOCUS       sequence-308581-        7804 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven multiplexed hybrid guide
            (hg)RNA targeting TK1 using SpCas9 and four intergenic sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7804)
  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 7804)
  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_5
FEATURES             Location/Qualifiers
     source          1..7804
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     368..387
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     621..638
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    755..776
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(791..796,797..814,815..821)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    829..845
                     /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     834..856
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     853..869
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     853..869
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     888..908
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        890..908
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        936..1162
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1163..1343
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1390..1515
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2008..2241
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2426..2470
                     /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(2989..3237)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(3047..3066)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(3217..3237)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    3279..3396
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3445..3955
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3874..3892
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3977..4576
                     /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(3977..3996)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4473..4493
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4592..5180
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4645..4665)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5063..5074)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5252..5485
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5563..5684
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5600..5619)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5654..5673
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5724..5859
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5879..5898)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5880..5898)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5908..5925)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5908..5924)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5917..5939)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6066..6521
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6153..6172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6363..6384
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6547..6651
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6652..6720,6721..7512)
                     /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(6870..6889)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7683..7804,1..467)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
       61 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
      121 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
      181 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
      241 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
      301 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
      361 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
      421 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
      481 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
      541 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
      601 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac
      661 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
      721 ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg
      781 caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat
      841 aacaatttca cacaggaaac agctatgacc atgattacgc caagcgcgca attaaccctc
      901 actaaaggga acaaaagctg gagctgcaag cttaatgtag tcttatgcaa tactcttgta
      961 gtcttgcaac atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg
     1021 tgcatgccga ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac
     1081 gggtctgaca tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa
     1141 gtgcctagct cgatacataa acgggtctct ctggttagac cagatctgag cctgggagct
     1201 ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca
     1261 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta
     1321 gtcagtgtgg aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa
     1381 ccagaggagc tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag
     1441 gggcggcgac tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat
     1501 gggtgcgaga gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg
     1561 ttaaggccag ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag
     1621 ctagaacgat tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata
     1681 ctgggacagc tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat
     1741 acagtagcaa ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct
     1801 ttagacaaga tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct
     1861 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
     1921 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
     1981 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
     2041 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
     2101 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     2161 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     2221 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     2281 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     2341 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     2401 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     2461 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     2521 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     2581 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg
     2641 acccagagaa ttcaaaaaaa actctgtgtg ctcaaggggg gctaatctac acttagtaga
     2701 aattacaata aagcatgaca atcagtatat ctacacttag tagaaattag gagaggctgg
     2761 caatattaaa ctatctacac ttagtagaaa ttagaactgg ttattgttca gtcattatct
     2821 acacttagta gaaattattg tggggttgtt tgttaggctc atctacactt agtagaaatt
     2881 agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatt tcaacttgct
     2941 atgctgtttc cagcatagct ctgaaactcg tcgatgccta tgacagccgg tacctcgtcc
     3001 tttccacaag atatataaag ccaagaaatc gaaatacttt caagttacgg taagcatatg
     3061 atagtccatt ttaaaacata attttaaaac tgcaaactac ccaagaaatt attactttct
     3121 acgtcacgta ttttgtacta atatctttgt gtttacagtc aaattaattc taattatctc
     3181 tctaacagcc ttgtatcgta tatgcaaata tgaaggaatc atgggaaata ggccctcgaa
     3241 ttctagatct tgagacaaat ggcagtattc atccacaatt ttaaaagaaa aggggggatt
     3301 ggggggtaca gtgcagggga aagaatagta gacataatag caacagacat acaaactaaa
     3361 gaattacaaa aacaaattac aaaaattcaa aattttcggg tttattacag ggacagcaga
     3421 gatccacttt ggcgccggct cgagggggcc cgggttgcgc cttttccaag gcagccctgg
     3481 gtttgcgcag ggacgcggct gctctgggcg tggttccggg aaacgcagcg gcgccgaccc
     3541 tgggtctcgc acattcttca cgtccgttcg cagcgtcacc cggatcttcg ccgctaccct
     3601 tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt cgggaaggtt ccttgcggtt
     3661 cgcggcgtgc cggacgtgac aaacggaagc cgcagctcac actagtaccc tcgcagacgg
     3721 acagcgccag ggagcaatgg cagcgcgccg accgcgatgg gctgtggcca atagcggctg
     3781 ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg gtgcgggagg cggggtgtgg
     3841 ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc gcattctgca agcctccgga
     3901 gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc gacctctctc cccaggggga
     3961 tccaccggag cttaccatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
     4021 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     4081 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     4141 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg ccgtggcggt
     4201 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     4261 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     4321 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggtgtttcgc ccgaccacca
     4381 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     4441 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     4501 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     4561 caagcccggt gcctgaacgc gttaagtcga caatcaacct ctggattaca aaatttgtga
     4621 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     4681 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     4741 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     4801 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     4861 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     4921 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     4981 ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg
     5041 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     5101 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     5161 cctttgggcc gcctccccgc gtcgacttta agaccaatga cttacaaggc agctgtagat
     5221 cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga
     5281 caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     5341 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     5401 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     5461 tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt catgtcatct tattattcag
     5521 tatttataac ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct
     5581 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     5641 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag
     5701 ctatcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     5761 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg
     5821 agctattcca gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc
     5881 ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga
     5941 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
     6001 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
     6061 atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     6121 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     6181 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     6241 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     6301 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     6361 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     6421 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     6481 atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga
     6541 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     6601 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     6661 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     6721 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     6781 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     6841 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac
     6901 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     6961 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     7021 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     7081 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     7141 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca
     7201 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     7261 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     7321 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     7381 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     7441 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     7501 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     7561 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     7621 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     7681 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     7741 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     7801 ttca
//
