LOCUS       sequence-223912-        8239 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses mEGFP-VPS4A in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8239)
  AUTHORS   Bleck M, Itano MS, Johnson DS, Thomas VK, North AJ, Bieniasz PD, 
            Simon SM
  TITLE     Temporal and spatial organization of ESCRT protein recruitment 
            during HIV-1 budding.
  JOURNAL   Proc Natl Acad Sci U S A. 2014 Aug 19;111(33):12211-6. doi: 
            10.1073/pnas.1321655111. Epub 2014 Aug 6.
  PUBMED    25099357
REFERENCE   2  (bases 1 to 8239)
  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: 
            "10.1073/pnas.1321655111"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2014-08-19- 19"; volume: "111"; issue: "33"; pages: "12211-6"
COMMENT     Sequence Label: pLNCX2-mEGFP-VPS4A
FEATURES             Location/Qualifiers
     source          1..8239
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             414..589
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    652..851
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1052..1468
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1398..1420
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1436..1452
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             1512..2306
                     /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(1566..1585)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2176..2195
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     enhancer        2377..2680
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2681..2884
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2835..2855
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2929..2948
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2929..2947
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2975..2984
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2981..2983,2984..2986,2987..3697)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3026..3047)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3287..3306)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3634..3655
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             5141..5734
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5837..5859)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5945..6085
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6000..6019
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6100..6117)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6271..6859)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6351..6370)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7030..7821,7822..7890))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7653..7672
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7891..7995)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8063..8081
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tttgaaagac cccacccgta ggtggcaagc tagcttaagt aacgccactt tgcaaggcat
       61 ggaaaaatac ataactgaga atagaaaagt tcagatcaag gtcaggaaca aagaaacagc
      121 tgaataccaa acaggatatc tgtggtaagc ggttcctgcc ccggctcagg gccaagaaca
      181 gatgagacag ctgagtgatg ggccaaacag gatatctgtg gtaagcagtt cctgccccgg
      241 ctcggggcca agaacagatg gtccccagat gcggtccagc cctcagcagt ttctagtgaa
      301 tcatcagatg tttccagggt gccccaagga cctgaaaatg accctgtacc ttatttgaac
      361 taaccaatca gttcgcttct cgcttctgtt cgcgcgcttc cgctctccga gctcaataaa
      421 agagcccaca acccctcact cggcgcgcca gtcttccgat agactgcgtc gcccgggtac
      481 ccgtattccc aataaagcct cttgctgttt gcatccgaat cgtggtctcg ctgttccttg
      541 ggagggtctc ctctgagtga ttgactaccc acgacggggg tctttcattt gggggctcgt
      601 ccgggatttg gagacccctg cccagggacc accgacccac caccgggagg taagctggcc
      661 agcaacttat ctgtgtctgt ccgattgtct agtgtctatg tttgatgtta tgcgcctgcg
      721 tctgtactag ttagctaact agctctgtat ctggcggacc cgtggtggaa ctgacgagtt
      781 ctgaacaccc ggccgcaacc ctgggagacg tcccagggac tttgggggcc gtttttgtgg
      841 cccgacctga ggaagggagt cgatgtggaa tccgaccccg tcaggatatg tggttctggt
      901 aggagacgag aacctaaaac agttcccgcc tccgtctgaa tttttgcttt cggtttggaa
      961 ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct
     1021 gactgtgttt ctgtatttgt ctgaaaatta gggccagact gttaccactc ccttaagttt
     1081 gaccttaggt cactggaaag atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa
     1141 gaagagacgt tgggttacct tctgctctgc agaatggcca acctttaacg tcggatggcc
     1201 gcgagacggc acctttaacc gagacctcat cacccaggtt aagatcaagg tcttttcacc
     1261 tggcccgcat ggacacccag accaggtccc ctacatcgtg acctgggaag ccttggcttt
     1321 tgacccccct ccctgggtca agccctttgt acaccctaag cctccgcctc ctcttcctcc
     1381 atccgccccg tctctccccc ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta
     1441 tccagccctc actccttctc taggcgccgg aattccgatc tgatcaagag acaggatgag
     1501 gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc gcttgggtgg
     1561 agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat gccgccgtgt
     1621 tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg tccggtgccc
     1681 tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt
     1741 gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta ttgggcgaag
     1801 tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta tccatcatgg
     1861 ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc gaccaccaag
     1921 cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc gatcaggatg
     1981 atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc
     2041 gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca
     2101 tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt gtggcggacc
     2161 gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc ggcgaatggg
     2221 ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc atcgccttct
     2281 atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaatagt aatcaattac
     2341 ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
     2401 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
     2461 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
     2521 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
     2581 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
     2641 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
     2701 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
     2761 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
     2821 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
     2881 agctctctgg ctaactagag aacccactgc ttactggctt atcgaaatta atacgactca
     2941 ctatagggag acccaagctc tagcgctacc ggtcgccacc atggtgagca agggcgagga
     3001 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     3061 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     3121 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     3181 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     3241 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     3301 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     3361 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     3421 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     3481 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     3541 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa
     3601 gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     3661 cgccgggatc actctcggca tggacgagct gtacaagtcc ggactcagat ccccggaatt
     3721 catgacaacg tcaaccctcc agaaagccat tgatctggtg acgaaagcca cagaggagga
     3781 caaagccaag aactacgagg aggcgctgcg gctgtaccag catgcggtgg agtacttcct
     3841 ccacgctatc aagtatgagg cccacagcga caaggccaag gagagcattc gagccaagtg
     3901 cgtgcagtac ctagaccggg ccgagaagct gaaggattat ttacgaagca aagagaaaca
     3961 cggcaagaag ccagtcaaag agaaccagag tgagggcaag ggcagtgaca gtgacagtga
     4021 aggggataat ccggagaaaa agaaactgca agaacagttg atgggtgccg tcgtgatgga
     4081 gaagcccaac atacggtgga acgacgtggc cgggctggag ggggccaagg aggccctcaa
     4141 agaagctgtc attttgccaa tcaaattccc acacttgttc acaggcaagc gcaccccctg
     4201 gcgggggatt ctgctgttcg gaccccctgg cacagggaaa tcctacctgg ccaaagccgt
     4261 ggcaacagag gccaacaact ccaccttctt ctctgtgtcc tcctcagatc tgatgtccaa
     4321 gtggctgggg gagagtgaaa agctggtcaa gaacctgttt gagctggcca ggcagcacaa
     4381 gccctccatc atcttcatcg atgaggtgga ttccctctgc gggtcccgaa atgaaaatga
     4441 gagtgaggcc gcccggagga tcaaaacgga gttcttggtc cagatgcagg gggtggggaa
     4501 taacaatgat gggactctgg ttcttggagc cacaaacatc ccatgggtgt tggattcggc
     4561 catcaggagg aggtttgaaa aacgaattta tatccccttg ccggaggaag ctgcccgcgc
     4621 ccagatgttc cggttgcatc tcgggagcac tccccacaac ctcacggatg caaacatcca
     4681 cgagctggcc cggaagacgg aaggctactc gggcgcggac atcagcatca tcgtgcggga
     4741 ctctctcatg cagcccgtga ggaaggtgca gtcggccaca cacttcaaaa aggtctgtgg
     4801 cccctctcgc accaacccca gcatgatgat tgatgacctc ctgactccat gctcaccagg
     4861 ggacccagga gccatggaga tgacttggat ggatgtccct ggggacaaac tcttagagcc
     4921 tgtggtttgc atgtcggaca tgctgcggtc tctggccacc acccggccca cggtgaatgc
     4981 agacgacctc ctgaaagtga agaaattctc agaggacttt gggcaagaga gttaactcga
     5041 gctcaagctt gtttggccga ggcggccgct tgtcgacagg ccttaatggc ctaacatcga
     5101 taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta
     5161 ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaaat acataactga
     5221 gaatagagaa gttcagatca aggtcaggaa cagatggaac agctgaatat gggccaaaca
     5281 ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggaacagctg
     5341 aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa
     5401 cagatggtcc ccagatgcgg tccagccctc agcagtttct agagaaccat cagatgtttc
     5461 cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg
     5521 cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc
     5581 tcactcgggg cgccagtcct ccgattgact gagtcgcccg ggtacccgtg tatccaataa
     5641 accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga
     5701 gtgattgact acccgtcagc gggggtcttt catttggggg ctcgtccggg atcgggagac
     5761 ccctgcccag ggaccaccga cccaccaccg ggaggtaagc tggctgcctc gcgcgtttcg
     5821 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     5881 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     5941 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc ttaactatgc
     6001 ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg
     6061 cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6121 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6181 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6241 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6301 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6361 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6421 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6481 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6541 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6601 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6661 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     6721 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6781 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6841 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6901 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6961 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     7021 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7081 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7141 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7201 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7261 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7321 tttgcgcaac gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat
     7381 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7441 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7501 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7561 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7621 accgagttgc tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt
     7681 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7741 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7801 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7861 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7921 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7981 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     8041 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaagaatt
     8101 cataccagat caccgaaaac tgtcctccaa atgtgtcccc ctcacactcc caaattcgcg
     8161 ggcttctgcc tcttagacca ctctacccta ttccccacac tcaccggagc caaagccgcg
     8221 gcccttccgt ttctttgct
//
