LOCUS       sequence-81801-e        8852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral vector for enhanced transgene and 
            eGFP-coexpression in primary B cells and B-cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8852)
  AUTHORS   Mock U, Thiele R, Uhde A, Fehse B, Horn S
  TITLE     Efficient lentiviral transduction and transgene expression in 
            primary human B cells.
  JOURNAL   Hum Gene Ther Methods. 2012 Dec;23(6):408-15. doi: 
            10.1089/hgtb.2012.160.
  PUBMED    23240650
REFERENCE   2  (bases 1 to 8852)
  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: "10.1089/hgtb.2012";
            journalName: "Hum Gene Ther Methods"; date: "2012-12"; volume: "23";
            issue: "6"; pages: "408-15"
COMMENT     Sequence Label: LeGO-hEµMAR-iG2
FEATURES             Location/Qualifiers
     source          1..8852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1686..1919
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2104..2148
                     /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    2446..2563
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     2622..2638
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(2680..2713)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3810..4216
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     misc_feature    join(4299..4873,4874..4876,4877..4885)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4466..4483)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4693..4712
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(4886..4888,4889..4891,4892..5605)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4886..4888,4889..4891,4892..5602)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4931..4952)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5192..5211)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5539..5560
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(5652..5685)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5741..6329
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5794..5814)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6212..6223)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6332..6348)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     LTR             6858..7038
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7100..7688)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7180..7199)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7859..8650,8651..8719))
                     /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     8482..8501
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8720..8824)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgcgctga tcttcagacc tggaggagga
     1561 gatatgaggg acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca
     1621 ttaggagtag cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg
     1681 ggaataggag ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcg
     1741 tcaatgacgc tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac
     1801 aatttgctga gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc
     1861 aagcagctcc aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg
     1921 gggatttggg gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt
     1981 tggagtaata aatctctgga acagatttgg aatcacacga cctggatgga gtgggacaga
     2041 gaaattaaca attacacaag cttaatacac tccttaattg aagaatcgca aaaccagcaa
     2101 gaaaagaatg aacaagaatt attggaatta gataaatggg caagtttgtg gaattggttt
     2161 aacataacaa attggctgtg gtatataaaa ttattcataa tgatagtagg aggcttggta
     2221 ggtttaagaa tagtttttgc tgtactttct atagtgaata gagttaggca gggatattca
     2281 ccattatcgt ttcagaccca cctcccaacc ccgaggggac ccgacaggcc cgaaggaata
     2341 gaagaagaag gtggagagag agacagagac agatccattc gattagtgaa cggatcggca
     2401 ctgcgtgcgc caattctgca gacaaatggc agtattcatc cacaatttta aaagaaaagg
     2461 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     2521 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     2581 cagcagagat ccagtttggt tagtaccggg cccgctctag tcgaggtcga cggtatcgat
     2641 aagctcgctt cacgagattc cagcaggtcg agggacctaa taacttcgta tagcatacat
     2701 tatacgaagt tatattaagg gttccaagct taagcggccg gccgctgaaa gaccccacct
     2761 gtaggtttgg caagctagca ggattgttta tcttaggagg catgcttact gttaaaagac
     2821 aggatatgtt tgaagtggct tctgagaaaa atggttaaga aaattatgac ttaaaaatgt
     2881 gagagatttt caagtatatt aattttttta actgtccaag tatttgaaat tcttatcatt
     2941 tgattaacac ccatgagtga tatgtgtctg gaattgaggc caaagcaagc tcagctaaga
     3001 aatactagca cagtgctgtc ggccccgatg cgggactgcg ttttgaccat cataaatcaa
     3061 gtttattttt ttaattaatt gagcgaagct ggaagcagat gatgaattag agtcaagatg
     3121 gctgcatggg ggtctccggc acccacagca ggtggcagga agcaggtcac cgcgagagtc
     3181 tattttagga agcaaaaaaa cacaattggt aaatttatca cttctggttg tgaagaggtg
     3241 gttttgccca ggcccagatc tgaaagtgct ctactgagca aaacaacacc tggacaattt
     3301 gcgtttctaa aataaggcga ggctgaccga aactgaaaag gcttttttta actatctgaa
     3361 tttcatttcc aatcttagct tatcaactgc tagtttgtgc aaacagcata tcaacttcta
     3421 aactgcattc atttttaaag taagatgttt aagaaattaa acagtcttag ggagagttta
     3481 tgactgtatt caaaaagttt tttaaattag cttgttatcc cttcatgtga taactaatct
     3541 caaatacttt ttcgatacct cagagcatta ttttcataat gactgtgttc acaatctttt
     3601 taggttaact cgttttctct ttgtgattaa ggagaaacac tttgatattc tgatagagtg
     3661 gccttcattt tagtattttt caagaccact tttcaactac tcactttagg ataagtttta
     3721 ggtaaaatgt gcatcattat cctgaattat ttcagttaag catgttagtt ggtggcataa
     3781 gagaaaactc aatcagatag ctagctgcag taacgccatt ttgcaaggca tggaaaaata
     3841 ccaaaccaag aatagagaag ttcagatcaa gggcgggtac atgaaaatag ctaacgttgg
     3901 gccaaacagg atatctgcgg tgagcagttt cggccccggc ccggggccaa gaacagatgg
     3961 tcaccgcagt ttcggccccg gcccgaggcc aagaacagat ggtccccaga tatggcccaa
     4021 ccctcagcag tttcttaaga cccatcagat gtttccaggc tcccccaagg acctgaaatg
     4081 accctgcgcc ttatttgaat taaccaatca gcctgcttct cgcttctgtt cgcgcgcttc
     4141 tgcttcccga gctctataaa agagctcaca acccctcact cggcgcgcca gtcctccgat
     4201 tgactgagtc gcccggatcc cagtgtggtg gtacgggaat tcctgcaggc ctcgagggcc
     4261 ggcgcgccgc ggccgctacg taaattccgc cccccccccc cctctccctc ccccccccct
     4321 aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt
     4381 tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg
     4441 acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc
     4501 gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt
     4561 tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta
     4621 taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg
     4681 gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag
     4741 gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac atgtgtttag
     4801 tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa
     4861 acacgatgat aatatggcca caaccatggt gagcaagggc gaggagctgt tcaccggggt
     4921 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     4981 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     5041 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     5101 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     5161 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     5221 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     5281 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     5341 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     5401 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     5461 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     5521 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     5581 cggcatggac gagctgtaca agtaaagcgg ccggccgcca gcacagtggt cgaaattcgt
     5641 cgagggacct aataacttcg tatagcatac attatacgaa gttatacatg tttaagggtt
     5701 ccggttccac taggtacaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa
     5761 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     5821 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     5881 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     5941 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     6001 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     6061 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     6121 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     6181 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     6241 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     6301 tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgat cgagacctag
     6361 aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc
     6421 tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac
     6481 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     6541 aagggctaat tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac
     6601 aaggctactt ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga
     6661 cctttggatg gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg
     6721 aaggagagaa cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga
     6781 gagaagtatt agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc
     6841 tgcatccgga ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     6901 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     6961 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     7021 tgtggaaaat ctctagcagc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     7081 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     7141 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     7201 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     7261 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     7321 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     7381 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     7441 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     7501 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     7561 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     7621 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     7681 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     7741 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     7801 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     7861 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     7921 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     7981 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     8041 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     8101 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     8161 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     8221 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     8281 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     8341 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     8401 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     8461 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     8521 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     8581 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     8641 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     8701 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     8761 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     8821 cgcgcacatt tccccgaaaa gtgccacctg ac
//
