LOCUS       sequence-308523-        7672 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral plasmid for expressing U6 driven multiplexed hybrid guide
            (hg)RNA targeting TK1 using SpCas9 and an intergenic site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7672)
  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 7672)
  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_2
FEATURES             Location/Qualifiers
     source          1..7672
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    29..154
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    647..880
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1065..1109
                     /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(1496..1744)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1554..1573)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1724..1744)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     misc_feature    1786..1903
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1952..2462
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2381..2399
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2484..3083
                     /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(2484..2503)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2980..3000
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3099..3687
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3152..3172)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3570..3581)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3759..3992
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4070..4191
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4107..4126)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4161..4180
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4231..4366
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4386..4405)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4387..4405)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4415..4432)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4415..4431)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4424..4446)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4573..5028
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4660..4679)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4870..4891
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5054..5158
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5159..5227,5228..6019)
                     /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(5377..5396)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6190..6778
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6679..6698
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6932..6949
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7066..7087
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7102..7107,7108..7125,7126..7132)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7140..7156
                     /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     7145..7167
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7164..7180
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7164..7180
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7199..7219
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7201..7219
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        7247..7473
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             7474..7654
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
ORIGIN
        1 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
       61 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
      121 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
      181 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
      241 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
      301 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
      361 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
      421 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
      481 cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg gacaattgga
      541 gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta gcacccacca
      601 aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga gctttgttcc
      661 ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg ctgacggtac
      721 aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg agggctattg
      781 aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc caggcaagaa
      841 tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg ggttgctctg
      901 gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat aaatctctgg
      961 aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac aattacacaa
     1021 gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat gaacaagaat
     1081 tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca aattggctgt
     1141 ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga atagtttttg
     1201 ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg tttcagaccc
     1261 acctcccaac cccgagggga cccagagaat tcaaaaaaaa ctctgtgtgc tcaagggggg
     1321 ctaatctaca cttagtagaa attattgtgg ggttgtttgt taggctcatc tacacttagt
     1381 agaaattagc accgactcgg tgccactttt tcaagttgat aacggactag ccttatttca
     1441 acttgctatg ctgtttccag catagctctg aaactcgtcg atgcctatga cagccggtac
     1501 ctcgtccttt ccacaagata tataaagcca agaaatcgaa atactttcaa gttacggtaa
     1561 gcatatgata gtccatttta aaacataatt ttaaaactgc aaactaccca agaaattatt
     1621 actttctacg tcacgtattt tgtactaata tctttgtgtt tacagtcaaa ttaattctaa
     1681 ttatctctct aacagccttg tatcgtatat gcaaatatga aggaatcatg ggaaataggc
     1741 cctcgaattc tagatcttga gacaaatggc agtattcatc cacaatttta aaagaaaagg
     1801 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     1861 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     1921 cagcagagat ccactttggc gccggctcga gggggcccgg gttgcgcctt ttccaaggca
     1981 gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg
     2041 ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg
     2101 ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct
     2161 tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc agctcacact agtaccctcg
     2221 cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata
     2281 gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg
     2341 ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc
     2401 ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc
     2461 agggggatcc accggagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     2521 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     2581 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     2641 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgccg
     2701 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     2761 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     2821 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggt gtttcgcccg
     2881 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     2941 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     3001 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     3061 tgacccgcaa gcccggtgcc tgaacgcgtt aagtcgacaa tcaacctctg gattacaaaa
     3121 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     3181 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     3241 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     3301 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     3361 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3421 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3481 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     3541 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     3601 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     3661 ggatctccct ttgggccgcc tccccgcgtc gactttaaga ccaatgactt acaaggcagc
     3721 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     3781 acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc agatctgagc
     3841 ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa gcttgccttg
     3901 agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag
     3961 acccttttag tcagtgtgga aaatctctag cagtacgtat agtagttcat gtcatcttat
     4021 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat
     4081 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     4141 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     4201 gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4261 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4321 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg gacgtaccca
     4381 attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta caacgtcgtg
     4441 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     4501 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     4561 atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4621 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4681 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4741 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     4801 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4861 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     4921 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     4981 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag gtggcacttt
     5041 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta
     5101 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat
     5161 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt
     5221 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg
     5281 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga
     5341 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg
     5401 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt
     5461 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg
     5521 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg
     5581 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga
     5641 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc
     5701 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc
     5761 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc
     5821 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg
     5881 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac
     5941 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc
     6001 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt
     6061 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     6121 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     6181 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     6241 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     6301 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     6361 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     6421 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     6481 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
     6541 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     6601 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     6661 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     6721 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     6781 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     6841 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
     6901 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
     6961 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
     7021 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
     7081 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
     7141 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcgcgc
     7201 aattaaccct cactaaaggg aacaaaagct ggagctgcaa gcttaatgta gtcttatgca
     7261 atactcttgt agtcttgcaa catggtaacg atgagttagc aacatgcctt acaaggagag
     7321 aaaaagcacc gtgcatgccg attggtggaa gtaaggtggt acgatcgtgc cttattagga
     7381 aggcaacaga cgggtctgac atggattgga cgaaccactg aattgccgca ttgcagagat
     7441 attgtattta agtgcctagc tcgatacata aacgggtctc tctggttaga ccagatctga
     7501 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
     7561 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     7621 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg ga
//
