LOCUS       sequence-322009-        7856 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  The empty pLenti backbone with multiple cloning site that allows 
            shuttling between pMY and pMX vectors .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7856)
  AUTHORS   van der Donk LEH, van der Spek J, van Duivenvoorde T, Ten Brink MS, 
            Geijtenbeek TBH, Kuijl CP, van Heijst JWJ, Ates LS
  TITLE     An optimized retroviral toolbox for overexpression and genetic 
            perturbation of primary lymphocytes.
  JOURNAL   Biol Open. 2022 Feb 15;11(2):bio059032. doi: 10.1242/bio.059032. 
            Epub 2022 Mar 1.
  PUBMED    35229875
REFERENCE   2  (bases 1 to 7856)
  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"; journalName: "Biol 
            Open."; date: "2022-02-15"; volume: "11(2):bio059032. doi"; pages: "
            10.1242/bio.059032. Epub 2022 Mar 1"
COMMENT     Sequence Label: pLenti-EFS-MCS-WPRE
FEATURES             Location/Qualifiers
     source          1..7856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..199
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     154..174
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             217..397
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    444..569
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1066..1299
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1484..1528
                     /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"
     misc_feature    1826..1943
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2007..2218
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     misc_feature    2327..2915
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2380..2400)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2798..2809)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             2987..3220
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     primer_bind     complement(3246..3263)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3252..3476
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3522..3950
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3609..3628)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3819..3840
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3959..3979)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3964..4293
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4144..4279
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4206..4225
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4341..4388
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4407..4781
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    4911..5032
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4948..4967)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5002..5021
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5081..5097)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5081..5097)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5094..5116)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5105..5121
                     /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)."
     promoter        complement(join(5129..5135,5136..5153,5154..5159))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5174..5195
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5312..5329)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5483..6071)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5563..5582)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6242..7033,7034..7102))
                     /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     6865..6884
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7103..7207)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7282..7301)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7476..7855
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
       61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
      121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
      181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc
      241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
      301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
      361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga
      421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca
      481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct
      541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat
      601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat
      661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag
      721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta
      781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag
      841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac
      901 agcaagcggc cggccgctga tcttcagacc tggaggagga gatatgaggg acaattggag
      961 aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag cacccaccaa
     1021 ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag ctttgttcct
     1081 tgggttcttg ggagcagcag gaagcactat gggcgcagcg tcaatgacgc tgacggtaca
     1141 ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga gggctattga
     1201 ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc aggcaagaat
     1261 cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg gttgctctgg
     1321 aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata aatctctgga
     1381 acagatttgg aatcacacga cctggatgga gtgggacaga gaaattaaca attacacaag
     1441 cttaatacac tccttaattg aagaatcgca aaaccagcaa gaaaagaatg aacaagaatt
     1501 attggaatta gataaatggg caagtttgtg gaattggttt aacataacaa attggctgtg
     1561 gtatataaaa ttattcataa tgatagtagg aggcttggta ggtttaagaa tagtttttgc
     1621 tgtactttct atagtgaata gagttaggca gggatattca ccattatcgt ttcagaccca
     1681 cctcccaacc ccgaggggac ccgacaggcc cgaaggaata gaagaagaag gtggagagag
     1741 agacagagac agatccattc gattagtgaa cggatcggca ctgcgtgcgc caattctgca
     1801 gacaaatggc agtattcatc cacaatttta aaagaaaagg ggggattggg gggtacagtg
     1861 caggggaaag aatagtagac ataatagcaa cagacataca aactaaagaa ttacaaaaac
     1921 aaattacaaa aattcaaaat tttcgggttt attacaggga cagcagagat ccagtttggt
     1981 taattaaggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag tccccgagaa
     2041 gttgggggga ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg
     2101 ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag aaccgtatat
     2161 aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca gaacacaggg
     2221 gatccggtcg tgcggccgca cggaaacgcg tggcctggca tgccgcgacc ctgcaggttt
     2281 ctgaagcttg agtacatcga tacggaaacg cgcgcgttaa gtcgacaatc aacctctgga
     2341 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     2401 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     2461 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     2521 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     2581 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     2641 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     2701 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     2761 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     2821 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     2881 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac
     2941 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     3001 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag
     3061 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc
     3121 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga
     3181 tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg
     3241 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     3301 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     3361 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     3421 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     3481 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     3541 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     3601 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3661 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3721 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     3781 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     3841 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     3901 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     3961 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     4021 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     4081 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     4141 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     4201 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     4261 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     4321 ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat
     4381 aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca
     4441 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg
     4501 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg
     4561 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg
     4621 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc
     4681 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg
     4741 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg
     4801 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct
     4861 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta
     4921 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     4981 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     5041 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt
     5101 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag
     5161 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt
     5221 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
     5281 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     5341 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5401 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5461 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5521 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5581 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5641 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5701 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5761 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5821 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5881 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5941 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6001 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6061 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6121 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6181 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6241 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     6301 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6361 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6421 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6481 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6541 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6601 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6661 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6721 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6781 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6841 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6901 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6961 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7021 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7081 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7141 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7201 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga
     7261 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct
     7321 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac
     7381 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct
     7441 gcttcgcgat gtacgggcca gatatacgcc gcgttgacat tgattattga ctagttatta
     7501 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
     7561 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
     7621 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
     7681 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     7741 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
     7801 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatgg
//
