LOCUS       sequence-220248-        8240 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 8240)
  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 8240)
  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..8240
                     /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        2378..2681
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2682..2885
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2836..2856
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2930..2949
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2930..2948
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      2976..2985
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2982..2984,2985..2987,2988..3698)
                     /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(3027..3048)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3288..3307)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3635..3656
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             5142..5735
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5838..5860)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5946..6086
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6001..6020
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6101..6118)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6272..6860)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6352..6371)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7031..7822,7823..7891))
                     /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     7654..7673
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7892..7996)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8064..8082
                     /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 ttcgtaatag taatcaatta
     2341 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     2401 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     2461 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     2521 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     2581 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     2641 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     2701 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     2761 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     2821 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     2881 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     2941 actataggga gacccaagct ctagcgctac cggtcgccac catggtgagc aagggcgagg
     3001 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     3061 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     3121 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     3181 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3241 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3301 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     3361 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     3421 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     3481 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     3541 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca
     3601 agctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     3661 ccgccgggat cactctcggc atggacgagc tgtacaagtc cggactcaga tccccggaat
     3721 tcatgacaac gtcaaccctc cagaaagcca ttgatctggt gacgaaagcc acagaggagg
     3781 acaaagccaa gaactacgag gaggcgctgc ggctgtacca gcatgcggtg gagtacttcc
     3841 tccacgctat caagtatgag gcccacagcg acaaggccaa ggagagcatt cgagccaagt
     3901 gcgtgcagta cctagaccgg gccgagaagc tgaaggatta tttacgaagc aaagagaaac
     3961 acggcaagaa gccagtcaaa gagaaccaga gtgagggcaa gggcagtgac agtgacagtg
     4021 aaggggataa tccggagaaa aagaaactgc aagaacagtt gatgggtgcc gtcgtgatgg
     4081 agaagcccaa catacggtgg aacgacgtgg ccgggctgga gggggccaag gaggccctca
     4141 aagaagctgt cattttgcca atcaaattcc cacacttgtt cacaggcaag cgcaccccct
     4201 ggcgggggat tctgctgttc ggaccccctg gcacagggaa atcctacctg gccaaagccg
     4261 tggcaacaga ggccaacaac tccaccttct tctctgtgtc ctcctcagat ctgatgtcca
     4321 agtggctggg ggagagtgaa aagctggtca agaacctgtt tgagctggcc aggcagcaca
     4381 agccctccat catcttcatc gatgaggtgg attccctctg cgggtcccga aatgaaaatg
     4441 agagtgaggc cgcccggagg atcaaaacgg agttcttggt ccagatgcag ggggtgggga
     4501 ataacaatga tgggactctg gttcttggag ccacaaacat cccatgggtg ttggattcgg
     4561 ccatcaggag gaggtttgaa aaacgaattt atatcccctt gccggaggaa gctgcccgcg
     4621 cccagatgtt ccggttgcat ctcgggagca ctccccacaa cctcacggat gcaaacatcc
     4681 acgagctggc ccggaagacg gaaggctact cgggcgcgga catcagcatc atcgtgcggg
     4741 actctctcat gcagcccgtg aggaaggtgc agtcggccac acacttcaaa aaggtctgtg
     4801 gcccctctcg caccaacccc agcatgatga ttgatgacct cctgactcca tgctcaccag
     4861 gggacccagg agccatggag atgacttgga tggatgtccc tggggacaaa ctcttagagc
     4921 ctgtggtttg catgtcggac atgctgcggt ctctggccac cacccggccc acggtgaatg
     4981 cagacgacct cctgaaagtg aagaaattct cagaggactt tgggcaagag agttaactcg
     5041 agctcaagct tgtttggccg aggcggccgc ttgtcgacag gccttaatgg cctaacatcg
     5101 ataaaataaa agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt
     5161 aggtttggca agctagctta agtaacgcca ttttgcaagg catggaaaaa tacataactg
     5221 agaatagaga agttcagatc aaggtcagga acagatggaa cagctgaata tgggccaaac
     5281 aggatatctg tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggaacagct
     5341 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
     5401 acagatggtc cccagatgcg gtccagccct cagcagtttc tagagaacca tcagatgttt
     5461 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
     5521 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
     5581 ctcactcggg gcgccagtcc tccgattgac tgagtcgccc gggtacccgt gtatccaata
     5641 aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg
     5701 agtgattgac tacccgtcag cgggggtctt tcatttgggg gctcgtccgg gatcgggaga
     5761 cccctgccca gggaccaccg acccaccacc gggaggtaag ctggctgcct cgcgcgtttc
     5821 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     5881 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     5941 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg
     6001 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat
     6061 gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc
     6121 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     6181 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     6241 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6301 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6361 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6421 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6481 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6541 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6601 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6661 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     6721 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6781 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6841 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6901 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6961 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     7021 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7081 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7141 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7201 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7261 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7321 gtttgcgcaa cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta
     7381 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7441 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7501 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7561 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7621 gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt
     7681 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7741 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7801 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7861 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7921 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7981 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta
     8041 ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat
     8101 tcataccaga tcaccgaaaa ctgtcctcca aatgtgtccc cctcacactc ccaaattcgc
     8161 gggcttctgc ctcttagacc actctaccct attccccaca ctcaccggag ccaaagccgc
     8221 ggcccttccg tttctttgct
//
