LOCUS       sequence-308580-        7716 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven multiplexed hybrid guide
            (hg)RNA targeting TK1 using SpCas9 and two intergenic sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7716)
  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 7716)
  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_3
FEATURES             Location/Qualifiers
     source          1..7716
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     76..93
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    210..231
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(246..251,252..269,270..276)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    284..300
                     /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     289..311
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     308..324
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     308..324
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     343..363
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        345..363
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        391..617
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             618..798
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    845..970
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1463..1696
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1881..1925
                     /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(2356..2604)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2414..2433)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2584..2604)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    2646..2763
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2812..3322
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3241..3259
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3344..3943
                     /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(3344..3363)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3840..3860
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3959..4547
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4012..4032)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4430..4441)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4619..4852
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4930..5051
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4967..4986)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5021..5040
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5091..5226
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5246..5265)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5247..5265)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5275..5292)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5275..5291)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5284..5306)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5433..5888
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5520..5539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5730..5751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5914..6018
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6019..6087,6088..6879)
                     /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(6237..6256)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7050..7638
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7539..7558
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
       61 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa
      121 ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag gtttcccgac
      181 tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca ttaggcaccc
      241 caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag cggataacaa
      301 tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa ccctcactaa
      361 agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc ttgtagtctt
      421 gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat
      481 gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc
      541 tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc
      601 tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg
      661 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
      721 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
      781 tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga
      841 ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg
      901 gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg
      961 cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag
     1021 gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga
     1081 acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg
     1141 acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt
     1201 agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga
     1261 caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct
     1321 tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat aaatataaag
     1381 tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga
     1441 gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa
     1501 gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta
     1561 tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac
     1621 tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa
     1681 aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg
     1741 tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct
     1801 ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag
     1861 aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa
     1921 gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga
     1981 tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag
     2041 ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg aggggaccca
     2101 gagaattcaa aaaaaactct gtgtgctcaa ggggggctaa tctacactta gtagaaatta
     2161 gaactggtta ttgttcagtc attatctaca cttagtagaa attattgtgg ggttgtttgt
     2221 taggctcatc tacacttagt agaaattagc accgactcgg tgccactttt tcaagttgat
     2281 aacggactag ccttatttca acttgctatg ctgtttccag catagctctg aaactcgtcg
     2341 atgcctatga cagccggtac ctcgtccttt ccacaagata tataaagcca agaaatcgaa
     2401 atactttcaa gttacggtaa gcatatgata gtccatttta aaacataatt ttaaaactgc
     2461 aaactaccca agaaattatt actttctacg tcacgtattt tgtactaata tctttgtgtt
     2521 tacagtcaaa ttaattctaa ttatctctct aacagccttg tatcgtatat gcaaatatga
     2581 aggaatcatg ggaaataggc cctcgaattc tagatcttga gacaaatggc agtattcatc
     2641 cacaatttta aaagaaaagg ggggattggg gggtacagtg caggggaaag aatagtagac
     2701 ataatagcaa cagacataca aactaaagaa ttacaaaaac aaattacaaa aattcaaaat
     2761 tttcgggttt attacaggga cagcagagat ccactttggc gccggctcga gggggcccgg
     2821 gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg
     2881 ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag
     2941 cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc
     3001 cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc
     3061 agctcacact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc
     3121 gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga
     3181 aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg
     3241 gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg
     3301 aatcaccgac ctctctcccc agggggatcc accggagctt accatgaccg agtacaagcc
     3361 cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc
     3421 gttcgccgac taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt
     3481 caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt
     3541 cgcggacgac ggcgccgccg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc
     3601 ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca
     3661 gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc
     3721 caccgtcggt gtttcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc
     3781 cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg
     3841 caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga
     3901 aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa
     3961 tcaacctctg gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc
     4021 ttttacgcta tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat
     4081 ggctttcatt ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg
     4141 gcccgttgtc aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg
     4201 ttggggcatt gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat
     4261 tgccacggcg gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt
     4321 gggcactgac aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc
     4381 ctgtgttgcc acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa
     4441 tccagcggac cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg
     4501 ccttcgccct cagacgagtc ggatctccct ttgggccgcc tccccgcgtc gactttaaga
     4561 ccaatgactt acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg
     4621 gaagggctaa ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc
     4681 tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag
     4741 cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct
     4801 ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtacgtat
     4861 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
     4921 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4981 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     5041 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc
     5101 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     5161 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     5221 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     5281 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     5341 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     5401 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     5461 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     5521 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     5581 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     5641 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     5701 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     5761 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     5821 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     5881 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5941 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     6001 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     6061 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     6121 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     6181 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     6241 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     6301 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     6361 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     6421 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6481 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6541 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6601 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6661 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6721 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6781 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6841 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6901 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6961 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     7021 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     7081 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     7141 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     7201 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     7261 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     7321 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     7381 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     7441 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7501 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7561 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7621 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7681 tggccttttg ctcacatgtt ctttcctgcg ttatcc
//
