LOCUS       sequence-337630-        7529 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Replacing the RSV promoter of pLKO.1 with the CMV promoter improves 
            lentiviral production with third-generation viral packaging system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7529)
  AUTHORS   Lee S, Cobrinik D
  TITLE     Improved third-generation lentiviral packaging with pLKO.1C vectors.
  JOURNAL   Biotechniques. 2020 Jun;68(6):349-352. doi: 10.2144/btn-2019-0155. 
            Epub 2020 Mar 6.
  PUBMED    32141762
REFERENCE   2  (bases 1 to 7529)
  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: 
            "10.2144/btn-2019-0155"; journalName: "Biotechniques"; date: 
            "2020-06"; volume: "68"; issue: "6"; pages: "349-352"
COMMENT     Sequence Label: pLKO.1C-shSCR
FEATURES             Location/Qualifiers
     source          1..7529
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     66..84
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             169..768
                     /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(169..188)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     665..685
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             896..1129
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1201..1322
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1238..1257)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1292..1311
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1362..1497
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1517..1536)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1518..1536)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1546..1563)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1546..1562)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1555..1577)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1704..2159
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1791..1810)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2001..2022
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2185..2289
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2290..2358,2359..3150)
                     /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(2508..2527)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3321..3909
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3810..3829
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4063..4080
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4197..4218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4233..4238,4239..4256,4257..4263)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4271..4287
                     /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     4276..4298
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4295..4311
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4295..4311
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4330..4350
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4332..4350
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     enhancer        4451..4830
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4831..5034
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4985..5005
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5049..5229
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5276..5401
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5894..6127
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6312..6356
                     /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        6654..6902
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     6654..6674
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     6825..6844
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    7000..7117
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        join(7166..7529,1..147)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
ORIGIN
        1 cgggaagggg cggtgcggga ggcggggtgt ggggcggtag tgtgggccct gttcctgccc
       61 gcgcggtgtt ccgcattctg caagcctccg gagcgcacgt cggcagtcgg ctccctcgtt
      121 gaccgaatca ccgacctctc tccccagggg gatccaccgg agcttaccat gaccgagtac
      181 aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc
      241 gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag
      301 cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg
      361 tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg
      421 ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc
      481 gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc
      541 ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg
      601 ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg
      661 ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg
      721 cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg cccgccccac
      781 gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcggtacc tttaagacca
      841 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
      901 gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg
      961 ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct
     1021 caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt
     1081 aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca
     1141 tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg
     1201 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     1261 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     1321 tatcatgtct ggctctagct atcccgcccc taactccgcc catcccgccc ctaactccgc
     1381 ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg
     1441 aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag
     1501 ggacgtaccc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt
     1561 acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc
     1621 ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt
     1681 gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1741 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1801 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1861 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1921 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1981 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2041 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2101 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     2161 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2221 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     2281 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     2341 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     2401 ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt
     2461 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     2521 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     2581 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     2641 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     2701 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     2761 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     2821 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     2881 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     2941 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     3001 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     3061 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     3121 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     3181 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     3241 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     3301 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     3361 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     3421 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     3481 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     3541 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     3601 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     3661 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3721 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3781 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3841 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3901 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3961 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     4021 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     4081 gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     4141 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     4201 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
     4261 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
     4321 gccaagcgcg caattaaccc tcactaaagg gaacaaaagc tggagctgca agcttggcca
     4381 ttgcatacgt tgtatccata tcataatatg tacatttata ttggctcatg tccaacatta
     4441 ccgccatgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     4501 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     4561 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     4621 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     4681 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     4741 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     4801 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     4861 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     4921 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     4981 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta
     5041 gtgaaccggg gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag
     5101 ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc
     5161 gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa
     5221 tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag aggagctctc
     5281 tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt
     5341 gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt
     5401 cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa ggccaggggg
     5461 aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag aacgattcgc
     5521 agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg gacagctaca
     5581 accatccctt cagacaggat cagaagaact tagatcatta tataatacag tagcaaccct
     5641 ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag acaagataga
     5701 ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccgctgatc ttcagacctg
     5761 gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa
     5821 ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa
     5881 gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg
     5941 gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc
     6001 agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct
     6061 ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac
     6121 agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga
     6181 atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt
     6241 gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa
     6301 accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga
     6361 attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag
     6421 gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg
     6481 gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg
     6541 aaggaataga agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg
     6601 gatctcgacg gtatcgatca cgagactagc ctcgagcggc cgcccccttc accgagggcc
     6661 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
     6721 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
     6781 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
     6841 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     6901 ccggtcctaa ggttaagtcg ccctcgctcg agcgagggcg acttaacctt aggtttttga
     6961 attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
     7021 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     7081 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     7141 agatccactt tggccgcggc tcgagggggt tggggttgcg ccttttccaa ggcagccctg
     7201 ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc
     7261 ctgggtctcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc
     7321 ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt
     7381 tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc ctcgcagacg
     7441 gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct
     7501 gctcagcagg gcgcgccgag agcagcggc
//
