LOCUS       sequence-348382-        8323 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  V5 tagged Vps53 is expressed in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8323)
  AUTHORS   Perez-Victoria FJ, Mardones GA, Bonifacino JS
  TITLE     Requirement of the human GARP complex for mannose 
            6-phosphate-receptor-dependent sorting of cathepsin D to lysosomes.
  JOURNAL   Mol Biol Cell. 2008 Jun;19(6):2350-62. doi: 10.1091/mbc.e07-11-1189.
            Epub 2008 Mar 26.
  PUBMED    18367545
REFERENCE   2  (bases 1 to 8323)
  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: 
            "10.1091/mbc.e07-11-1189"; journalName: "Mol Biol Cell"; date: 
            "2008-06"; volume: "19"; issue: "6"; pages: "2350-62"
COMMENT     Sequence Label: pEF6 Vps53-V5-6His
FEATURES             Location/Qualifiers
     source          1..8323
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        194..1372
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          425..1363
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1320..1340
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1389..1408
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1389..1407
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4042..4083
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4093..4110
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4133..4150)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4139..4363
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4409..4837
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4496..4515)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4706..4727
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4846..4866)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4851..5180
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5031..5166
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5093..5112
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5228..5275
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5294..5692
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    5850..5971
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5887..5906)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5941..5960
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6020..6036)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6020..6036)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6033..6055)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6044..6060
                     /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(6068..6074,6075..6092,6093..6098))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6113..6134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6251..6268)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6411..6999)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6491..6510)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7170..7961,7962..8030))
                     /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     7793..7812
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8031..8135)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8210..8229)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
       61 ggccagatat acgcgttgac attgattatt gactaggctt ttgcaaaaag ctttgcaaag
      121 atggataaag ttttaaacag agaggaatct ttgcagctaa tggaccttct aggtcttgaa
      181 aggagtggga attggctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc
      241 cgagaagttg gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt
      301 aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc
      361 gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac
      421 acaggtaagt gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg
      481 cgtgccttga attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt
      541 tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg
      601 agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc
      661 ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac
      721 gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc
      781 ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag
      841 gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc
      901 tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc
      961 ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag
     1021 ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa
     1081 aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc
     1141 caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg
     1201 gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg
     1261 gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct
     1321 caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggaattag
     1381 cttggtacta atacgactca ctatagggag acccaagctg gctaggtaag cttggtaccg
     1441 agctcggatc cactagtcca gtgtggtgga attgccctta tgatggagga ggaggaactg
     1501 gagttcgtgg aggagctgga ggctgtgctg cagctcacgc cagaggtgca actcgccatc
     1561 gagcaggtat tcccaagcca ggatcctctt gatcaagctg acttcaatgc cgttgagtat
     1621 atcaatactc tgttcccaac agagcaatct ctggcaaaca ttgacgatgt tgtgaacaaa
     1681 attagactga aaataaggag actggatgac aatatccgga ctgtcgtaag aggtcagaca
     1741 aatatggggc aggatggaag gcaagcactt gaagaggccc agaaagccat tcagcaactc
     1801 tttggcaaaa tcaaggatat caaagacaaa gcagaaaagt cagaacaaat ggtgaaagag
     1861 atcacccgag atattaagca gttagatcac gccaagcgcc acctgaccac ctcaatcacg
     1921 accctgaacc acctgcacat gctggcaggc ggggtggact ccctggaagc catgaccagg
     1981 cgcagacagt atggagaagt tgctaatctc cttcagggtg taatgaatgt tctggagcac
     2041 tttcacaaat acatgggaat cccacagatc cgacagcttt ctgaaagggt gaaggctgca
     2101 cagacagagt taggacagca aatcctggct gactttgaag aagcgtttcc ttcgcagggt
     2161 accaagaggc cgggaggacc cagcaatgtc ctgagagatg cgtgcctggt ggctaatatt
     2221 ttggacccca gaatcaaaca agaaatcatc aaaaagttta ttaaacagca tctatcagag
     2281 tatctagtac tttttcaaga aaaccaagat gtcgcctggc tggacaaaat tgacagacgt
     2341 tatgcttggg tgaaacgcca gcttgtggac tatgaagaga agtatggccg gatgtttcct
     2401 cgggagtggt gcatgacgga gaggatctca gtagagtttt gccacgtgac gagggcagaa
     2461 ctttccaaaa tcatgcgtgc cagagctaag gaaattgaag tgaaattgct tctttttgcg
     2521 atccagagaa caaccaactt tgaggggttt cttgcaaaac gcttctccgg ttgcaccctg
     2581 acagatggaa ccctgaaaaa gctggagtcc ccaccaccat ctaccaatcc tttcctggaa
     2641 gatgagacaa caccagagat ggaggagctg gctctggaga aaggagagtt agagcaaccg
     2701 aagaagccca aggccccgga caacccgttc cacggcatcg tttccaagtg ttttgagcct
     2761 catctctacg tgtacattga gtcccaggac aaaaacctca gcgagctgat agaccggttt
     2821 gtggcagatt tcaaagccca gggcccaccc aagcccaaca ctgacgaagg gggtgctgtg
     2881 ctccccagct gcgctgacct ctttgtctac tacaagaagt gcatggtgca gtgctcacag
     2941 ctcagcacag gcgagcccat gatcgccctg actaccatct tccagaagta cctccgagaa
     3001 tacgcctgga aaatcctctc tggcaacctg cctaaaacca gcagcagcag tggggggctg
     3061 accatcagca gcctcctcaa agagaaggag ggctcagaag tggccaggtt cactctggag
     3121 gagctgtgtc tcatctgcag catcctgagc acagccgagt attgtctggc taccacccag
     3181 cagctagaag aaaaactcaa agaaaaagtt gatgtgagcc tgactgaacg aattaatctc
     3241 acaggagaaa tggacacatt cagcactgtc atctccagca gcatccagct gttggttcaa
     3301 gatctggatg cagcctgtga ccctgccctg attgccatga gcaagatgcc ttggcagaac
     3361 gtggagcatg ttggtgacca gagcccttat gtcacttctg ttattcttca cattaagcaa
     3421 aacgtcccca tcatccgtga caacctggct tccacacgga aatacttcac acagttctgc
     3481 attaaatttg caaattcgtt cattcccaaa ttcatcaccc acctcttcaa gtgtaagccg
     3541 atcagcatgg tgggagcaga gcagcttctg ctggacaccc actccctgaa gatggtcctg
     3601 ctcgaccttc cgtccattgg ctcccaggtg gtgaggaagg cccctgccag ttacactaag
     3661 attgtcgtga aaggcatgac ccgggctgaa atgatcctta aggtggtgat ggcccctcat
     3721 gaaccgttgg tggtatttgt tgacaactac atcaaactcc ttacggactg caactcagaa
     3781 accttccaga aaatactgga tatgaagggg ctgaagagga gtgagcagag cagcatgctg
     3841 gaacttttgc ggcagaggct ccctgccccg ccctcgggga cagaaggttc cagcaccctg
     3901 tcgctgattg ccccaacacc ggagcaggag tcatcccgaa tccgcaaact ggagaaactg
     3961 attaaaaaga gactgaaggg caattctgca gatatccagc acagtggcgg ccgctcgagt
     4021 ctagagggcc cgcggttcga aggtaagcct atccctaacc ctctcctcgg tctcgattct
     4081 acgcgtaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact
     4141 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     4201 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     4261 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     4321 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     4381 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     4441 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     4501 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     4561 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     4621 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     4681 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     4741 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     4801 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     4861 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     4921 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4981 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     5041 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     5101 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     5161 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     5221 agcacgtgtt gacaattaat catcggcata gtatatcggc atagtataat acgacaaggt
     5281 gaggaactaa accatggcca agcctttgtc tcaagaagaa tccaccctca ttgaaagagc
     5341 aacggctaca atcaacagca tccccatctc tgaagactac agcgtcgcca gcgcagctct
     5401 ctctagcgac ggccgcatct tcactggtgt caatgtatat cattttactg ggggaccttg
     5461 tgcagaactc gtggtgctgg gcactgctgc tgctgcggca gctggcaacc tgacttgtat
     5521 cgtcgcgatc ggaaatgaga acaggggcat cttgagcccc tgcggacggt gccgacaggt
     5581 gcttctcgat ctgcatcctg ggatcaaagc catagtgaag gacagtgatg gacagccgac
     5641 ggcagttggg attcgtgaat tgctgccctc tggttatgtg tgggagggct aagcacttcg
     5701 tggccgagga gcaggactga cacgtgctac gagatttcga ttccaccgcc gccttctatg
     5761 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     5821 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     5881 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5941 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     6001 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     6061 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     6121 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     6181 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     6241 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     6301 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     6361 acatgtgagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     6421 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     6481 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     6541 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     6601 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     6661 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     6721 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     6781 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     6841 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     6901 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     6961 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     7021 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     7081 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     7141 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     7201 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     7261 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     7321 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     7381 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     7441 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     7501 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     7561 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     7621 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     7681 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     7741 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     7801 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     7861 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     7921 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     7981 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     8041 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     8101 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     8161 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     8221 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     8281 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gac
//
