LOCUS       sequence-256457-        7855 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lentiviral destination vector with Ef1a promoter and N-terminal V5 
            tag .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7855)
  TITLE     Barrett lab unpublished plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7855)
  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            "Unpublished"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_6.5_M2_Ef1a-V5-gateway
FEATURES             Location/Qualifiers
     source          1..7855
                     /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"
     regulatory      2391..2400
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2400..2441
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    2442..2566
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2591..2596,2597..2614,2615..2621)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2675..3334
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2741..2760)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3676..3981
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     3860..3879
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4022..4146)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     misc_feature    4281..4869
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4334..4354)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4752..4763)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             5059..5239
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        5308..5518
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5377..5512
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5628..5749
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     5719..5738
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5801..5818)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5972..6560)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6052..6071)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(6812..6851)
                     /label=fd terminator
                     /note="central terminator from bacteriophage fd (Otsuka and
                     Kunisawa, 1982)"
     CDS             complement(6861..7676)
                     /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(6861..7676)
                     /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     7584..7603
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     7668..7687
                     /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 cgcctatcga aattcactat agggagaccc aagctggcta gttaagcttc gccaccatgg
     2401 gtaagcctat ccctaaccct ctcctcggtc tcgattctac gacaagtttg tacaaaaaag
     2461 ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt ttgcataaaa
     2521 aacagactac ataatactgt aaaacacaac atatccagtc actatggcgg ccgcattagg
     2581 caccccaggc tttacacttt atgcttccgg ctcgtataat gtgtggattt tgagttagga
     2641 tccggcgaga ttttcaggag ctaaggaagc taaaatggag aaaaaaatca ctggatatac
     2701 caccgttgat atatcccaat ggcatcgtaa agaacatttt gaggcatttc agtcagttgc
     2761 tcaatgtacc tataaccaga ccgttcagct ggatattacg gcctttttaa agaccgtaaa
     2821 gaaaaataag cacaagtttt atccggcctt tattcacatt cttgcccgcc tgatgaatgc
     2881 tcatccggaa ttccgtatgg caatgaaaga cggtgagctg gtgatatggg atagtgttca
     2941 cccttgttac accgttttcc atgagcaaac tgaaacgttt tcatcgctct ggagtgaata
     3001 ccacgacgat ttccggcagt ttctacacat atattcgcaa gatgtggcgt gttacggtga
     3061 aaacctggcc tatttcccta aagggtttat tgagaatatg tttttcgtct cagccaatcc
     3121 ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat atggacaact tcttcgcccc
     3181 cgttttcacc atgggcaaat attatacgca aggcgacaag gtgctgatgc cgctggcgat
     3241 tcaggttcat catgccgtct gtgatggctt ccatgtcggc agaatgctta atgaattaca
     3301 acagtactgc gatgagtggc agggcggggc gtaaacgcgt ggatccggct tactaaaagc
     3361 cagataacag tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat
     3421 gtatacccga agtatgtcaa aaagaggtgt gctatgaagc agcgtattac agtgacagtt
     3481 gacagcgaca gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa
     3541 gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc
     3601 aggaagggat ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga
     3661 acagggactg gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt
     3721 ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc
     3781 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat
     3841 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt
     3901 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac
     3961 ctgatgttct ggggaatata aatgtcaggc tccgttatac acagccagtc tgcaggtcga
     4021 ccatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat
     4081 ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt tcttgtacaa
     4141 agtggtagta atgaccttag gagccaacag ctaagcacac acttcgaaag atatatttaa
     4201 atacacaatt aattaacaca aatacgcgta tctgcagtcg actttgccag accggagtcc
     4261 gaacacagct cttccagcat aatcaacctc tggattacaa aatttgtgaa agattgactg
     4321 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     4381 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     4441 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     4501 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     4561 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     4621 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     4681 cgtcctttcc ttggctgctc gcctgtgttg ccacctggat tctgcgcggg acgtccttct
     4741 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     4801 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     4861 cctccccgct tagtactggt acctttaaga ccaatgactt acaaggcagc tgtagatctt
     4921 agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa
     4981 gattccggaa tttatttgtg aaatttgtga tgctattgct ttatttgtaa accggtgcag
     5041 ctgctttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     5101 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     5161 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     5221 gtgtggaaaa tctctagcat ctagagtatg caaagcatgc atctcaatta gtcagcaacc
     5281 aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     5341 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     5401 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     5461 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggctag
     5521 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     5581 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     5641 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5701 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgct
     5761 agctgagcgg gctttttttt cttaggcctt cttccgcttc ctcgctcact gactcgctgc
     5821 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5881 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5941 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6001 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6061 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6121 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6181 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6241 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6301 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6361 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6421 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6481 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6541 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6601 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6661 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6721 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6781 gttcatccat agttgcctga ctcctgcgca gtccaaaaaa aaaggctcca aaaggagcct
     6841 ttaattgtat cggtgggccc ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta
     6901 ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa
     6961 aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc gattccgact
     7021 cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt atcaagtgag
     7081 aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagcttatg catttctttc
     7141 cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa
     7201 ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct gttaaaagga
     7261 caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata
     7321 ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc ggggatcgca
     7381 gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt cggaagaggc
     7441 ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt ggcaacgcta
     7501 cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa tcgatagatt
     7561 gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa atcagcatcc
     7621 atgttggaat ttaatcgcgg cctcgagcaa gacgtttccc gttgaatatg gctcataaca
     7681 ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgatga tatattttta
     7741 tcttgtgcaa tgtaacatca gagattttga gacacaacgt ggtttaaaca aatagtcaaa
     7801 agcctccggg cgactagtcg gggtcattag ttcatagccc atatatggag ttccg
//
