LOCUS       sequence-353682-        8731 bp    DNA     circular SYN 14-APR-2025
DEFINITION  V5 tagged Vps54 is expressed in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8731)
  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 8731)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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 Vps54-V5-6His
FEATURES             Location/Qualifiers
     source          1..8731
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(75..95)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        80..409
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      260..395
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     322..341
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        457..504
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             523..921
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    1079..1200
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1116..1135)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1170..1189
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1249..1265)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1249..1265)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1262..1284)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1273..1289
                     /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(1297..1303,1304..1321,1322..1327))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1342..1363
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1480..1497)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1640..2228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1720..1739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2399..3190,3191..3259))
                     /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     3022..3041
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3260..3364)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3439..3458)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3746..4924
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          3977..4915
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4872..4892
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     4941..4960
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4941..4959
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             8002..8043
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             8053..8070
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(8093..8110)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8099..8323
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(8369..8731,1..66)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8456..8475)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8666..8687
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg
       61 cgaattaatt ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc
      121 aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc
      181 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt
      241 cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc
      301 ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct
      361 attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg
      421 agcttgtata tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag
      481 tatatcggca tagtataata cgacaaggtg aggaactaaa ccatggccaa gcctttgtct
      541 caagaagaat ccaccctcat tgaaagagca acggctacaa tcaacagcat ccccatctct
      601 gaagactaca gcgtcgccag cgcagctctc tctagcgacg gccgcatctt cactggtgtc
      661 aatgtatatc attttactgg gggaccttgt gcagaactcg tggtgctggg cactgctgct
      721 gctgcggcag ctggcaacct gacttgtatc gtcgcgatcg gaaatgagaa caggggcatc
      781 ttgagcccct gcggacggtg ccgacaggtg cttctcgatc tgcatcctgg gatcaaagcc
      841 atagtgaagg acagtgatgg acagccgacg gcagttggga ttcgtgaatt gctgccctct
      901 ggttatgtgt gggagggcta agcacttcgt ggccgaggag caggactgac acgtgctacg
      961 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     1021 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     1081 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     1141 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     1201 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     1261 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     1321 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     1381 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     1441 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     1501 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     1561 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagg aaccgtaaaa
     1621 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     1681 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     1741 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     1801 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     1861 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     1921 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     1981 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     2041 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     2101 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     2161 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     2221 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     2281 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     2341 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     2401 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2461 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2521 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2581 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     2641 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     2701 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     2761 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     2821 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     2881 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     2941 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     3001 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     3061 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     3121 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     3181 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     3241 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     3301 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     3361 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     3421 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     3481 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     3541 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     3601 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactaggc ttttgcaaaa
     3661 agctttgcaa agatggataa agttttaaac agagaggaat ctttgcagct aatggacctt
     3721 ctaggtcttg aaaggagtgg gaattggctc cggtgcccgt cagtgggcag agcgcacatc
     3781 gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag
     3841 gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg
     3901 tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt
     3961 tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc tctttacggg
     4021 ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga ttcttgatcc
     4081 cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc
     4141 gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg
     4201 gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg
     4261 acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca
     4321 cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac
     4381 atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca
     4441 agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc
     4501 ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc
     4561 tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc
     4621 cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta
     4681 ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg
     4741 ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt
     4801 taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc
     4861 ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc
     4921 gtgaggaatt agcttggtac taatacgact cactataggg agacccaagc tggctaggta
     4981 agcttggtac cgagctcgga tccactagtc cagtgtggtg gaattgccct tgggggtacc
     5041 atggcttcaa gccacagttc ttctccagtg cctcaaggaa gcagcagtga tgttttcttt
     5101 aaaaaagagg tagatccaac aaaacacatt cgacctgtgc agtcactgcc agatgtgtgt
     5161 cccaaggaac ccacagttac agatcaacat agatggactg tgtatcattc caaagtaaat
     5221 ctcccagcag cattaaatga tcctacacta gcaaaaagag aatctgactt cttcacaaaa
     5281 acttggggat tggactttgt ggacactgaa gtcatacctt cactctacct cccacagatc
     5341 agcaaggaga attttatagc atatcagcag gagatctctc agagagagaa gattcatgag
     5401 agatgcaaga atatttgtcc tcctaaagat acctttgaaa ggactctttt acatactcat
     5461 gataaatcca ggacagatct ggagcaagta cctaagattt ttatgaaacc agattttgcc
     5521 ttggatgatt ctttaacttt taattcagtt ttaccatggt ctcattttaa tactgctggt
     5581 gggaaaggaa gtcgcgacgc agcttcttcc aagttgcttc aggaaaagct gagccattat
     5641 ctggatattg tggaagtaaa cattgctcac cagatctctc tacgttcaga agcatttttt
     5701 catgcaatga cctctcaaca cgaattgcag gactacctca agaaaactac ccaggctgta
     5761 aaaatgcttc gagataaaat tgcacagatt gataaagtta tgtgtgaagg atcactacag
     5821 attttaagac tggcacttac cagaaataat tgtgttaaag tatacaataa actgaagtta
     5881 atggccactg tacaccaaac tcagcctaca gtgcaggtgt tgttatctac ttctgaattt
     5941 gttggagcgt tggacttaat agcaacaact caagaggttc tgcagcagga gcttcagggc
     6001 attcacagct tccggcattt gggatcacag ctttgtgaat tagaaaagct gattgataaa
     6061 atgatgattg cggaattttc tacttattct catagtgact taaatagacc actggaaggt
     6121 gaatgccaag ttttagaaga ggaaagatta gtatctcttg tatttgggct tttgaaacaa
     6181 agaaaactta attttttaga aatctatggt gaagaaatga ttattacagc aaagaacatc
     6241 attaaagagc gtgtgattaa taaagtttca caaatagaag aaatagacac agacgttgtt
     6301 gtaaagcttg cagatcaaat gagaatgttg aattttcccc agtggattga tctgttaaag
     6361 gacattttct ctaagtttac agttttccta cagagagtta aggcaacatt gaatattatc
     6421 cacagtgttg ttctgtcagt tcttgagaaa agccaaagga ctagagaatt ggaagagatc
     6481 ccacagcaga ggagcgctgg gaaggacagc tccttggaca cagacgtggc ttacctgaca
     6541 cacgagggct ggttcataag tgatgctttc agtgagggtg agccagcatc tgcggcggtt
     6601 gatactacct ctcaaagaaa cacgtctcca catagcgagc cctgcagcag tgattcggta
     6661 tcagaaccag aatgtactac tgattcttca tccagcaagg agcagacatc agcgtgtgct
     6721 cctccaggag gtatagagat tatagtcagt gaagacatga ggttaactga cttggagcta
     6781 ggaaaactag caagtaacat ccaggagtta ctgtgtaatg cttcagatgt atgccatgat
     6841 cgagctgtca aatttctcat gtcaagagca aaggacggtt ttcttgaaaa attgaattcc
     6901 acggaattca tagcactctc gagattaatg gagacattca tagtggacac tgaacagatc
     6961 tgtgggagga agagcacgtc actgcttggg gcccttcaga gccaagcaaa taagtttgtg
     7021 aacaggtttc atgaagagag gcggaccaag ctcagcctcc tcttagacaa cgagcgttgg
     7081 aagcaggctg atgttcctgc agaatttcag gatcttgttg attctatagc ggatggaaag
     7141 attgctttac ctgaaaaaaa gccagtggtt acagaagaaa ggaagccagc tgatgttctt
     7201 gttgttgagg gacaccagta tgcagttgtc ggaaccgtct tgctgttaat aagaatcatt
     7261 cttgaatatt gccagtgtgt ggataacatc ccctccgtca ctactgacat gctcactcgc
     7321 ctgacagatc tgctgaagta cttcaattca agaagttgcc agttagttct tggagctggt
     7381 gctttgcaag ttgttggact aaaaacaatt actacaaaaa acttggccct ctcttcacgg
     7441 tgtctgcagt tgattgtgca ctacattccc gtgatccggg ctcactttga agcccgactg
     7501 ccccccaagc aatggagcat gcttagacat tttgatcaca tcacaaagga ttaccatgat
     7561 catatagctg aaatttcagc taagcttgta gcgataatgg atagcctatt tgataagctg
     7621 ttatcaaagt atgaggtgaa agctcctgtt ccttccccgt gtttcaggaa tatttgtaag
     7681 caaatgacaa aaatgcatga agctatattt gatcttcttc ccgaagaaca aacacagatg
     7741 ttatttttaa gaattaatgc aagttacaaa ctccacttga aaaaacagtt atcacactta
     7801 aatgtaataa atgacggagg acctcaaaat gggttggtca cagcagatgt agctttttac
     7861 actggaaatc ttcaagcctt aaaaggcctt aaagatctgg atctaaatat ggccgagatc
     7921 tgggaacaga agaggaaggg caattctgca gatatccagc acagtggcgg ccgctcgagt
     7981 ctagagggcc cgcggttcga aggtaagcct atccctaacc ctctcctcgg tctcgattct
     8041 acgcgtaccg gtcatcatca ccatcaccat tgagtttaaa cccgctgatc agcctcgact
     8101 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     8161 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     8221 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     8281 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     8341 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     8401 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     8461 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     8521 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     8581 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     8641 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     8701 cctatctcgg tctattcttt tgatttataa g
//
