LOCUS       sequence-177456-        8109 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses full length Flag-tagged ALIX with mutation I212D in 
            mammalian cells; internal ID WISP05-117.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8109)
  AUTHORS   Zhai Q, Landesman MB, Robinson H, Sundquist WI, Hill CP
  TITLE     Structure of the Bro1 domain protein BROX and functional analyses of
            the ALIX Bro1 domain in HIV-1 budding.
  JOURNAL   PLoS One. 2011;6(12):e27466. doi: 10.1371/journal.pone.0027466. Epub
            2011 Dec 5.
  PUBMED    22162750
REFERENCE   2  (bases 1 to 8109)
  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: "PLoS One.";
            date: "2011"; volume: "6(12):e27466. doi"; pages: " 
            10.1371/journal.pone.0027466. Epub 2011 Dec 5"
COMMENT     Sequence Label: pCINeoFlag ALIX; I212D
FEATURES             Location/Qualifiers
     source          1..8109
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        138..517
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        518..729
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     680..700
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     726..750
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          890..1022
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1005..1024
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        1067..1085
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1108..1131
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    3813..3934
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3824..3843)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3878..3897
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     rep_origin      4120..4575
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4207..4226)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4417..4438
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4692..5049
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4692..4883
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4715..4735)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4900..5035
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             5100..5894
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5154..5173)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5764..5783
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5958..6006
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(6047..6066)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6166..6188
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6226..6244)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6312..6416
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6417..6485,6486..7277)
                     /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     complement(6635..6654)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7448..8036
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7937..7956
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta
       61 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      121 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg
      181 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      241 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      301 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      361 ccacttggca gtacatcaag tgtatcatat cccaagtccg ccccctattg acgtcaatga
      421 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      481 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      541 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      601 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg
      661 cgatcgcccg ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata
      721 agcagagctc gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac
      781 agttaaattg ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt
      841 gactctctta aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa
      901 ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact
      961 cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac
     1021 aggtgtccac tcccagttca attacagctc ttaaggctag agtacttaat acgactcact
     1081 ataggctagc ctcgagaatt cgccatggat tacaaggatg acgacgataa gggccatatg
     1141 gcgacattca tctcggtgca gctgaaaaag acctcagagg tggacctggc caagccgctg
     1201 gtgaagttca tccagcagac ttacccaagc ggcggggaag agcaggccca gtactgccgc
     1261 gcggcggagg agctcagcaa gctgcgccgc gccgcagtcg gtcgtccgct ggacaagcac
     1321 gagggcgcgc tcgagacgct cctgagatat tatgatcaga tttgttctat tgaacccaaa
     1381 ttcccatttt ctgaaaatca gatctgcttg acatttacct ggaaggatgc tttcgataaa
     1441 ggttcacttt ttggaggctc tgtaaaactg gctcttgcaa gcttaggata tgaaaagagc
     1501 tgtgtgttgt tcaattgtgc agccttagct agccaaattg cagcagaaca gaacttagac
     1561 aacgatgaag gattgaaaat cgctgctaaa cattaccagt ttgctagtgg tgccttttta
     1621 catattaaag agacggtttt atctgcctta agtcgagagc cgaccgtgga catatctcca
     1681 gatactgttg ggaccctcag tcttattatg ctggcacagg ctcaagaagt atttttttta
     1741 aaagccacaa gagataaaat gaaagatgcc gacatagcta aattggctaa tcaggctgca
     1801 gattattttg gtgatgcttt caaacagtgt caatacaaag atactctccc caaggaggtg
     1861 ttccctgtct tggctgcaaa gcactgtatc atgcaggcca atgctgagta ccatcagtct
     1921 atcctggcaa aacagcagaa gaaatttgga gaagaaattg caaggttaca gcatgcagca
     1981 gaactgatta aaacagtggc atctcgctat gatgaatatg ttaatgtgaa ggatttttct
     2041 gacaaaatca atcgtgccct tgctgcagca aagaaggata atgacttcat ttatcatgat
     2101 cgagttccag accttaaaga tctagatcct attggcaaag ccacacttgt gaaatctacc
     2161 ccggtcaatg tacccatcag tcagaaattt actgatctgt ttgagaagat ggttcccgtg
     2221 tcagtacagc agtctttggc tgcctataat cagaggaaag ccgatttggt taacagatca
     2281 attgctcaga tgagagaagc caccactttg gcaaatgggg tgctagcttc ccttaatctt
     2341 ccagcagcaa ttgaagatgt gtctggagac actgtacctc agtctatatt gactaaatcc
     2401 agatctgtga ttgaacaggg aggcatccag actgttgatc agttgattaa agaactgcct
     2461 gaattactgc aacgaaatag agaaatccta gatgagtcat taaggttgtt ggatgaagaa
     2521 gaagcaaccg ataatgattt aagagcaaaa tttaaggaac gttggcaaag gacaccatcc
     2581 aatgaactgt ataagccttt aagagcagag ggaaccaact tcagaacagt tttagataaa
     2641 gctgtgcagg cagatggaca agtgaaagaa tgttaccagt ctcatcgtga caccatcgtg
     2701 cttttgtgta agccagagcc tgagctgaat gctgccatcc cttctgctaa tccagcaaag
     2761 accatgcagg gcagtgaggt tgtaaatgtc ttaaaatcct tattgtcaaa tcttgatgaa
     2821 gtaaagaagg aaagagaggg tctggagaat gacttgaaat ctgtgaattt tgacatgaca
     2881 agcaagtttt tgacagccct ggctcaagat ggtgtgataa atgaagaagc tctttctgtt
     2941 actgaactag atcgagtcta tggaggtctt acaactaaag tccaagaatc tctaaagaaa
     3001 caggagggac ttcttaaaaa tattcaggtc tcacatcagg aattttcaaa aatgaaacaa
     3061 tctaataatg aagctaactt aagagaagaa gttttgaaga atttagctac tgcgtatgac
     3121 aactttgttg aacttgtagc taatttgaag gaaggcacaa agttttacaa tgagttgact
     3181 gaaatcctgg tcaggttcca gaacaaatgc agtgatatag tttttgcacg gaagacagaa
     3241 agagatgaac tcttaaagga cttgcaacaa agcattgcca gagaacctag tgctccttca
     3301 attcctacac ctgcgtatca gtcctcacca gcaggaggac atgcaccaac tcctccaact
     3361 ccagcgccaa gaaccatgcc gcctactaag ccccagcccc cagccaggcc tccaccacct
     3421 gtgcttccag caaatcgagc tccttctgct actgctccat ctccagtggg ggctgggact
     3481 gctgcgccag ctccatcaca aacgcctggc tcagctcctc ctccacaggc gcagggacca
     3541 ccctatccca cctatccagg atatcctggg tattgccaaa tgcccatgcc catgggctat
     3601 aatccttatg cgtatggcca gtataatatg ccatatccac cagtgtatca ccagagtcct
     3661 ggacaggctc catacccggg accccagcag ccttcatacc ccttccctca gcccccacag
     3721 cagtcttact atccacagca gtaaggatcc tctagagtcg acccgggcgg ccgcttccct
     3781 ttagtgaggg ttaatgcttc gagcagacat gataagatac attgatgagt ttggacaaac
     3841 cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
     3901 atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca ttcattttat
     3961 gtttcaggtt cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg
     4021 tggtaaaatc cgataaggat cgatccgggc tggcgtaata gcgaagaggc ccgcaccgat
     4081 cgcccttccc aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat
     4141 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     4201 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     4261 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     4321 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     4381 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     4441 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     4501 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     4561 taacgcttac aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4621 ccgcatacgc ggatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg
     4681 ttaggtacct tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga
     4741 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     4801 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     4861 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     4921 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     4981 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     5041 ttttgcaaaa agcttgattc ttctgacaca acagtctcga acttaaggct agagccacca
     5101 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     5161 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     5221 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     5281 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     5341 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     5401 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     5461 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     5521 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     5581 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     5641 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     5701 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     5761 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     5821 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     5881 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     5941 gccatcacga tggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt
     6001 tgtgtgaatc gatagcgata aggatacgcg tatggtgcac tctcagtaca atctgctctg
     6061 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     6121 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     6181 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     6241 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     6301 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     6361 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     6421 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     6481 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     6541 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     6601 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     6661 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     6721 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     6781 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6841 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6901 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6961 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     7021 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     7081 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     7141 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     7201 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     7261 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     7321 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     7381 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     7441 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     7501 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     7561 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     7621 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     7681 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     7741 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     7801 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     7861 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     7921 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     7981 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     8041 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatggctc
     8101 gacagatct
//
