LOCUS       sequence-337632-        7582 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 7582)
  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 7582)
  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-YFP-shSCR
FEATURES             Location/Qualifiers
     source          1..7582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     132..151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     385..402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    519..540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(555..560,561..578,579..585)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    593..609
                     /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     598..620
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     617..633
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     617..633
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     652..672
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        654..672
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     enhancer        773..1152
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1153..1356
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1307..1327
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             1371..1551
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1598..1723
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2216..2449
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2634..2678
                     /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        2976..3224
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2976..2996
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3147..3166
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    3322..3439
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3488..3998
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3917..3935
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     regulatory      4008..4017
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4014..4016,4017..4019,4020..4733)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4059..4080)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4320..4339)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4667..4688
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4800..5033
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5105..5226
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5142..5161)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5196..5215
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5266..5401
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5421..5440)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5422..5440)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5450..5467)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5450..5466)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5459..5481)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5608..6063
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5695..5714)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5905..5926
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6089..6193
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6194..6262,6263..7054)
                     /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(6412..6431)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7225..7582,1..231)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
       61 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      121 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      181 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      241 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      301 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      361 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      421 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      481 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
      541 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      601 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
      661 cctcactaaa gggaacaaaa gctggagctg caagcttggc cattgcatac gttgtatcca
      721 tatcataata tgtacattta tattggctca tgtccaacat taccgccatg ttgacattga
      781 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
      841 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
      901 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
      961 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     1021 catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     1081 gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc
     1141 gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata gcggtttgac
     1201 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     1261 aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca aatgggcggt
     1321 aggcgtgtac ggtgggaggt ctatataagc agagctcgtt tagtgaaccg gggtctctct
     1381 ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc
     1441 ctcaataaag cttgccttga gtgcttcaag tagtgtgtgc ccgtctgttg tgtgactctg
     1501 gtaactagag atccctcaga cccttttagt cagtgtggaa aatctctagc agtggcgccc
     1561 gaacagggac ttgaaagcga aagggaaacc agaggagctc tctcgacgca ggactcggct
     1621 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
     1681 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
     1741 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
     1801 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
     1861 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
     1921 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
     1981 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
     2041 taagaccacc gcacagcaag cggccgctga tcttcagacc tggaggagga gatatgaggg
     2101 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     2161 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     2221 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc
     2281 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     2341 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     2401 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     2461 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     2521 aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca
     2581 attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg
     2641 aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa
     2701 attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa
     2761 tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt
     2821 ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag
     2881 gtggagagag agacagagac agatccattc gattagtgaa cggatctcga cggtatcgat
     2941 cacgagacta gcctcgagcg gccgccccct tcaccgaggg cctatttccc atgattcctt
     3001 catatttgca tatacgatac aaggctgtta gagagataat tggaattaat ttgactgtaa
     3061 acacaaagat attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg
     3121 cagttttaaa attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt
     3181 tcgatttctt ggctttatat atcttgtgga aaggacgaaa caccggtcct aaggttaagt
     3241 cgccctcgct cgagcgaggg cgacttaacc ttaggttttt gaattctcga cctcgagaca
     3301 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg
     3361 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat
     3421 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac tttggccgcg
     3481 gctcgagggg gttggggttg cgccttttcc aaggcagccc tgggtttgcg cagggacgcg
     3541 gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct cgcacattct
     3601 tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc cccccggcga
     3661 cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg tgccggacgt
     3721 gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc cagggagcaa
     3781 tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg
     3841 agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta gtgtgggccc
     3901 tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg
     3961 gctccctcgt tgaccgaatc accgacctct ctccccaggg ggatctcgcc accatggtga
     4021 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     4081 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     4141 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     4201 ccaccttcgg ctacggcctg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg
     4261 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     4321 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     4381 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     4441 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     4501 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     4561 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     4621 gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     4681 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taactttaag
     4741 accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact
     4801 ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct
     4861 ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa
     4921 gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc
     4981 tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag
     5041 ttcatgtcat cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga
     5101 gaggaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5161 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5221 atcttatcat gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact
     5281 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     5341 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     5401 ctagggacgt acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     5461 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     5521 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     5581 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     5641 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     5701 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     5761 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     5821 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5881 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     5941 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     6001 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     6061 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     6121 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     6181 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     6241 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     6301 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     6361 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     6421 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     6481 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     6541 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     6601 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     6661 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     6721 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     6781 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     6841 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     6901 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     6961 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     7021 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     7081 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     7141 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     7201 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     7261 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     7321 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     7381 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     7441 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     7501 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     7561 acagcccagc ttggagcgaa cg
//
