LOCUS       sequence-232789-        8811 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For LIC cloning with N-terminal V5 tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8811)
  AUTHORS   Cheng LC, Baboo S, Lindsay C, Brusman L, Martinez-Bartolome S, Tapia
            O, Zhang X, Yates JR 3rd, Gerace L
  TITLE     Identification of new transmembrane proteins concentrated at the 
            nuclear envelope using organellar proteomics of mesenchymal cells.
  JOURNAL   Nucleus. 2019 Dec;10(1):126-143. doi: 10.1080/19491034.2019.1618175.
  PUBMED    31142202
REFERENCE   2  (bases 1 to 8811)
  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: 
            "10.1080/19491034.2019"; journalName: "Nucleus"; date: "2019-12"; 
            volume: "10"; issue: "1"; pages: "126-143"
COMMENT     Sequence Label: pLV-EF1a-V5-LIC
FEATURES             Location/Qualifiers
     source          1..8811
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        78..289
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          290..1228
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1185..1205
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1244..1253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1253..1294
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     misc_feature    1462..2011
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1606..1623)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1833..1852
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2014..2613
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2014..2033)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2510..2530
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    2637..3225
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2690..2710)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3108..3119)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             3432..4065
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     primer_bind     complement(4193..4209)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4193..4209)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4206..4228)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4217..4233
                     /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(4241..4247,4248..4265,4266..4271))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4286..4307
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4424..4441)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4595..5183)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4675..4694)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5354..6145,6146..6214))
                     /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     5977..5996
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6215..6319)
                     /gene="bla"
                     /label=AmpR promoter
     polyA_signal    6367..6501
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6404..6423)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6458..6477
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     LTR             6669..7302
                     /label=3' LTR
                     /note="3' long terminal repeat (LTR) from HIV-1"
     misc_feature    7349..7474
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7971..8204
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             8389..8433
                     /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    8696..8811
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1 (lacking the first T)"
ORIGIN
        1 tcgggtttat tacagggaca gcagagatcc agtttatcga tgaggccctt tcgtcttcac
       61 tcgaggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg
      121 aggggtcggc aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga
      181 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt
      241 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt
      301 gtgtggttcc cgcgggcctg gcctctttac gggttatggc ccttgcgtgc cttgaattac
      361 ttccacctgg ctgcagtacg tgattcttga tcccgagctt cgggttggaa gtgggtggga
      421 gagttcgagg ccttgcgctt aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc
      481 tgggcgctgg ggccgccgcg tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt
      541 cgataagtct ctagccattt aaaatttttg atgacctgct gcgacgcttt ttttctggca
      601 agatagtctt gtaaatgcgg gccaagatct gcacactggt atttcggttt ttggggccgc
      661 gggcggcgac ggggcccgtg cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc
      721 gcggccaccg agaatcggac gggggtagtc tcaagctggc cggcctgctc tggtgcctgg
      781 cctcgcgccg ccgtgtatcg ccccgccctg ggcggcaagg ctggcccggt cggcaccagt
      841 tgcgtgagcg gaaagatggc cgcttcccgg ccctgctgca gggagctcaa aatggaggac
      901 gcggcgctcg ggagagcggg cgggtgagtc acccacacaa aggaaaaggg cctttccgtc
      961 ctcagccgtc gcttcatgtg actccacgga gtaccgggcg ccgtccaggc acctcgatta
     1021 gttctcgagc ttttggagta cgtcgtcttt aggttggggg gaggggtttt atgcgatgga
     1081 gtttccccac actgagtggg tggagactga agttaggcca gcttggcact tgatgtaatt
     1141 ctccttggaa tttgcccttt ttgagtttgg atcttggttc attctcaagc ctcagacagt
     1201 ggttcaaagt ttttttcttc catttcaggt gtcgtgagga tccgccgcca tgggtaagcc
     1261 tatccctaac cctctcctcg gtctcgattc tacggggggc ggagggggtg ggcccgggcc
     1321 gcgacgatgt ttaaacttcg aaacgcgtga attcctcgag ggcggccgct ctagagtcga
     1381 cgggccgcgg taacaattgt taactaactt aagctagcaa cggtttccct ctagcgggat
     1441 caattccgcc ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg
     1501 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga
     1561 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa
     1621 tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa
     1681 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct
     1741 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg
     1801 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg
     1861 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca
     1921 catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg
     1981 acgtggtttt cctttgaaaa acacgataat accatgaccg agtacaagcc cacggtgcgc
     2041 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     2101 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     2161 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     2221 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     2281 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     2341 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     2401 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     2461 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     2521 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     2581 acctggtgca tgacccgcaa gcccggtgcc tgacccgggc ggggcgcgtc tggaacaatc
     2641 aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt
     2701 ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg
     2761 ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc
     2821 ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt
     2881 ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg
     2941 ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg
     3001 gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg ctgctcgcct
     3061 gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc
     3121 cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc
     3181 ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgga attaattctg
     3241 cagtcgagac ctagaaaaac atggagcaat cacaagtagc aatacagcag ctaccaatgc
     3301 tgattgtgcc tggctagaag cacaagagga ggaggaggtg ggttttccag tcacacctca
     3361 ggtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga
     3421 aaagagggga ctggaagggc taattcactc ccaacgaaga caagatatcc ttgatctgtg
     3481 gatctaccac acacaaggct acttccctga ttagcagaac tacacaccag ggccaggggt
     3541 cagatatcca ctgacctttg gatggtgcta caagctagta ccagttgagc cagataaggt
     3601 agaagaggcc aataaaggag agaacaccag cttgttacac cctgtgagcc tgcatgggat
     3661 ggatgacccg gagagagaag tgttagagtg gaggtttgac agccgcctag catttcatca
     3721 cgtggcccga gagctgcatc cggagtactt caagaactgc tgatatcgag cttgctacaa
     3781 gggactttcc gctggggact ttccagggag gcgtggcctg ggcgggactg gggagtggcg
     3841 agccctcaga tcctgcatat aagcagctgc tttttgcctg tactgggtct ctctggttag
     3901 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat
     3961 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact
     4021 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc
     4081 atcttattat tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggcctt
     4141 gacattgcta gcgtttaccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     4201 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     4261 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     4321 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     4381 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     4441 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     4501 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     4561 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     4621 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     4681 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4741 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4801 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4861 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4921 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4981 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     5041 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     5101 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     5161 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     5221 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     5281 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     5341 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     5401 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     5461 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     5521 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     5581 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     5641 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     5701 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5761 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5821 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5881 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5941 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     6001 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     6061 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     6121 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     6181 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     6241 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     6301 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg
     6361 gagatcaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     6421 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     6481 gtatcttatc atgtctggat caactggata actcaagcta accaaaatca tcccaaactt
     6541 cccaccccat accctattac cactgccaat tacctgtggt ttcatttact ctaaacctgt
     6601 gattcctctg aattattttc attttaaaga aattgtattt gttaaatatg tactacaaac
     6661 ttagtagttg gaagggctaa ttcactccca aagaagacaa gatatccttg atctgtggat
     6721 ctaccacaca caaggctact tccctgatta gcagaactac acaccagggc caggggtcag
     6781 atatccactg acctttggat ggtgctacaa gctagtacca gttgagccag ataaggtaga
     6841 agaggccaat aaaggagaga acaccagctt gttacaccct gtgagcctgc atgggatgga
     6901 tgacccggag agagaagtgt tagagtggag gtttgacagc cgcctagcat ttcatcacgt
     6961 ggcccgagag ctgcatccgg agtacttcaa gaactgctga tatcgagctt gctacaaggg
     7021 actttccgct ggggactttc cagggaggcg tggcctgggc gggactgggg agtggcgagc
     7081 cctcagatcc tgcatataag cagctgcttt ttgcctgtac tgggtctctc tggttagacc
     7141 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     7201 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     7261 gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga
     7321 cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc ttgctgaagc
     7381 gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt tgactagcgg
     7441 aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga gaattagatc
     7501 gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat taaaacatat
     7561 agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt tagaaacatc
     7621 agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag gatcagaaga
     7681 acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa ggatagagat
     7741 aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa gtaagaccac
     7801 cgcacagcaa gcggccggcc gctgatcttc agacctggag gaggagatat gagggacaat
     7861 tggagaagtg aattatataa atataaagta gtaaaaattg aaccattagg agtagcaccc
     7921 accaaggcaa agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg
     7981 ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg
     8041 gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct
     8101 attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca
     8161 agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc
     8221 tctggaaaac tcatttgcac cactgctgtg ccttggaatg ctagttggag taataaatct
     8281 ctggaacaga tttggaatca cacgacctgg atggagtggg acagagaaat taacaattac
     8341 acaagcttaa tacactcctt aattgaagaa tcgcaaaacc agcaagaaaa gaatgaacaa
     8401 gaattattgg aattagataa atgggcaagt ttgtggaatt ggtttaacat aacaaattgg
     8461 ctgtggtata taaaattatt cataatgata gtaggaggct tggtaggttt aagaatagtt
     8521 tttgctgtac tttctatagt gaatagagtt aggcagggat attcaccatt atcgtttcag
     8581 acccacctcc caaccccgag gggacccgac aggcccgaag gaatagaaga agaaggtgga
     8641 gagagagaca gagacagatc cattcgatta gtgaacggat ctcgacggta tcgcctttaa
     8701 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     8761 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt t
//
