LOCUS       sequence-256467-        7544 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral expression vector with Ef1a promoter, EGFP gene, 
            C-terminal HA tag, SV40 promoter, and Zeo Resistance gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7544)
  TITLE     Barrett lab unpublished plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7544)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pSMPUW_6.19_Ex_Ef1a-EGFP-HA-SV40-Zeo
FEATURES             Location/Qualifiers
     source          1..7544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        305..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             522..702
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    749..873
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    908..1024
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1158..2336
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1389..2327
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2284..2304
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    2346..2370
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2391..2400
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2397..2399,2400..2402,2403..3113)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2442..2463)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2703..2722)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3050..3071
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(3115..3139)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3141..3167
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     promoter        3262..3472
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3284..3480
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3331..3466
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     regulatory      3517..3526
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3523..3897
                     /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"
     misc_feature    3970..4558
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4023..4043)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4441..4452)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4748..4928
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        4997..5207
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5066..5201
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5317..5438
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5408..5427
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5490..5507)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5661..6249)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5741..5760)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6501..6540)
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             complement(6550..7365)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(6550..7365)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     7273..7292
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7357..7376
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagagctgg tttagtgaac cgggtctctc tggttagacc
      541 agatttgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
      601 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
      661 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
      721 cctgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
      781 gcgcacggca agaggcgagg ggcggcgact gcagagtacg ccaaaatttt gactagcgga
      841 ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggaa aatagcggcc
      901 gccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
      961 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     1021 ttttcggggg atccgcggaa ttcgtccacg gtccaatctc ggtccgaacg agatatctgc
     1081 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct
     1141 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
     1201 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
     1261 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
     1321 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
     1381 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc
     1441 cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc
     1501 gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg
     1561 cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc
     1621 gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg
     1681 cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta
     1741 tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg
     1801 cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc
     1861 ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc
     1921 tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag
     1981 ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa
     2041 ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc
     2101 cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg
     2161 aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag
     2221 cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca
     2281 ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaggcg
     2341 cgccaacaag tttgtacaaa aaagcaggct aagctggcta gttaagcttc gccaccatgg
     2401 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2461 acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca
     2521 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2581 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2641 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2701 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2761 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2821 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2881 tcaaggccaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2941 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     3001 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     3061 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagcacccag
     3121 ctttcttgta caaagtggtt tacccatacg atgttccaga ttacgcttag taatgaacct
     3181 taggagccaa cagctaagca cacacttcga atgcatctca attagtcagc aaccaggtgt
     3241 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcgtct caattagtca
     3301 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3361 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3421 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3481 aagctttcta gaggtaccac cggagcttat ttaaatgcca ccatggccaa gttgaccagt
     3541 gccgttccgg tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg
     3601 ctcgggttct cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg
     3661 accctgttca tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg
     3721 tgggtgcgcg gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc
     3781 cgggacgcct ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc
     3841 gccctgcgcg acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactaatta
     3901 attaacacaa atacgcgtat ctgcagtcga ctttgccaga ccggagtccg aacacagctc
     3961 ttccagcata atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     4021 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     4081 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     4141 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     4201 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     4261 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     4321 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
     4381 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     4441 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     4501 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgctt
     4561 agtactggta cctttaagac caatgactta caaggcagct gtagatctta gccacttttt
     4621 aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag attccggaat
     4681 ttatttgtga aatttgtgat gctattgctt tatttgtaaa ccggtgcagc tgctttttgc
     4741 ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     4801 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     4861 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     4921 ctctagcatc tagagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa
     4981 gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac
     5041 catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     5101 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     5161 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctaga gatcataatc
     5221 agccatacca catttgtaga ggttttactt gctttaaaaa acctcccaca cctccccctg
     5281 aacctgaaac ataaaatgaa tgcaattgtt gttgttaact tgtttattgc agcttataat
     5341 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat
     5401 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgcta gctgagcggg
     5461 cttttttttc ttaggccttc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5521 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5581 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5641 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5701 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5761 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5821 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5881 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5941 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6001 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6061 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6121 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6181 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6241 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6301 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6361 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6421 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6481 gttgcctgac tcctgcgcag tccaaaaaaa aaggctccaa aaggagcctt taattgtatc
     6541 ggtgggccct tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
     6601 attatcaata ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag
     6661 gcagttccat aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
     6721 aatacaacct attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
     6781 agtgacgact gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc
     6841 aacaggccag ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
     6901 tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
     6961 aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga
     7021 atcaggatat tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
     7081 ccatgcatca tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
     7141 cagccagttt agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg
     7201 tttcagaaac aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
     7261 ttgcccgaca ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
     7321 taatcgcggc ctcgagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt
     7381 actgtttatg taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
     7441 gtaacatcag agattttgag acacaacgtg gtttaaacaa atagtcaaaa gcctccgggc
     7501 gactagtcgg ggtcattagt tcatagccca tatatggagt tccg
//
