LOCUS       sequence-223917-        8230 bp    DNA     circular SYN 14-APR-2025
DEFINITION  expresses mCherry-VPS4A in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8230)
  AUTHORS   Johnson DS, Bleck M, Simon SM
  TITLE     Timing of ESCRT-III protein recruitment and membrane scission during
            HIV-1 assembly.
  JOURNAL   Elife. 2018 Jul 4;7. pii: 36221. doi: 10.7554/eLife.36221.
  PUBMED    29972351
REFERENCE   2  (bases 1 to 8230)
  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"; journalName: "Elife."; 
            date: "2018-07-4"; volume: "7. pii: 36221. doi"; pages: " 
            10.7554/eLife.36221"
COMMENT     Sequence Label: pLNCX2-mCherry-VPS4A
FEATURES             Location/Qualifiers
     source          1..8230
                     /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             2981..3688
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(3126..3144)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3394..3413
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             5132..5725
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5828..5850)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5936..6076
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5991..6010
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6091..6108)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6262..6850)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6342..6361)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7021..7812,7813..7881))
                     /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     7644..7663
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7882..7986)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8054..8072
                     /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 ggataacatg gccatcatca aggagttcat gcgcttcaag gtgcacatgg agggctccgt
     3061 gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg agggcaccca
     3121 gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc ttcgcctggg acatcctgtc
     3181 ccctcagttc atgtacggct ccaaggccta cgtgaagcac cccgccgaca tccccgacta
     3241 cttgaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg
     3301 cggcgtggtg accgtgaccc aggactcctc cctgcaggac ggcgagttca tctacaaggt
     3361 gaagctgcgc ggcaccaact tcccctccga cggccccgta atgcagaaga agaccatggg
     3421 ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc gccctgaagg gcgagatcaa
     3481 gcagaggctg aagctgaagg acggcggcca ctacgacgct gaggtcaaga ccacctacaa
     3541 ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc aacatcaagt tggacatcac
     3601 ctcccacaac gaggactaca ccatcgtgga acagtacgaa cgcgccgagg gccgccactc
     3661 caccggcggc atggacgagc tgtacaagtc cggactcaga tccccggaat tcatgacaac
     3721 gtcaaccctc cagaaagcca ttgatctggt gacgaaagcc acagaggagg acaaagccaa
     3781 gaactacgag gaggcgctgc ggctgtacca gcatgcggtg gagtacttcc tccacgctat
     3841 caagtatgag gcccacagcg acaaggccaa ggagagcatt cgagccaagt gcgtgcagta
     3901 cctagaccgg gccgagaagc tgaaggatta tttacgaagc aaagagaaac acggcaagaa
     3961 gccagtcaaa gagaaccaga gtgagggcaa gggcagtgac agtgacagtg aaggggataa
     4021 tccggagaaa aagaaactgc aagaacagtt gatgggtgcc gtcgtgatgg agaagcccaa
     4081 catacggtgg aacgacgtgg ccgggctgga gggggccaag gaggccctca aagaagctgt
     4141 cattttgcca atcaaattcc cacacttgtt cacaggcaag cgcaccccct ggcgggggat
     4201 tctgctgttc ggaccccctg gcacagggaa atcctacctg gccaaagccg tggcaacaga
     4261 ggccaacaac tccaccttct tctctgtgtc ctcctcagat ctgatgtcca agtggctggg
     4321 ggagagtgaa aagctggtca agaacctgtt tgagctggcc aggcagcaca agccctccat
     4381 catcttcatc gatgaggtgg attccctctg cgggtcccga aatgaaaatg agagtgaggc
     4441 cgcccggagg atcaaaacgg agttcttggt ccagatgcag ggggtgggga ataacaatga
     4501 tgggactctg gttcttggag ccacaaacat cccatgggtg ttggattcgg ccatcaggag
     4561 gaggtttgaa aaacgaattt atatcccctt gccggaggaa gctgcccgcg cccagatgtt
     4621 ccggttgcat ctcgggagca ctccccacaa cctcacggat gcaaacatcc acgagctggc
     4681 ccggaagacg gaaggctact cgggcgcgga catcagcatc atcgtgcggg actctctcat
     4741 gcagcccgtg aggaaggtgc agtcggccac acacttcaaa aaggtctgtg gcccctctcg
     4801 caccaacccc agcatgatga ttgatgacct cctgactcca tgctcaccag gggacccagg
     4861 agccatggag atgacttgga tggatgtccc tggggacaaa ctcttagagc ctgtggtttg
     4921 catgtcggac atgctgcggt ctctggccac cacccggccc acggtgaatg cagacgacct
     4981 cctgaaagtg aagaaattct cagaggactt tgggcaagag agttaactcg agctcaagct
     5041 tgtttggccg aggcggccgc ttgtcgacag gccttaatgg cctaacatcg ataaaataaa
     5101 agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca
     5161 agctagctta agtaacgcca ttttgcaagg catggaaaaa tacataactg agaatagaga
     5221 agttcagatc aaggtcagga acagatggaa cagctgaata tgggccaaac aggatatctg
     5281 tggtaagcag ttcctgcccc ggctcagggc caagaacaga tggaacagct gaatatgggc
     5341 caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga acagatggtc
     5401 cccagatgcg gtccagccct cagcagtttc tagagaacca tcagatgttt ccagggtgcc
     5461 ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct
     5521 tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggg
     5581 gcgccagtcc tccgattgac tgagtcgccc gggtacccgt gtatccaata aaccctcttg
     5641 cagttgcatc cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac
     5701 tacccgtcag cgggggtctt tcatttgggg gctcgtccgg gatcgggaga cccctgccca
     5761 gggaccaccg acccaccacc gggaggtaag ctggctgcct cgcgcgtttc ggtgatgacg
     5821 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     5881 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     5941 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     6001 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     6061 aaaataccgc atcaggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     6121 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     6181 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     6241 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     6301 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     6361 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     6421 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     6481 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     6541 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     6601 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     6661 agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg
     6721 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     6781 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     6841 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     6901 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     6961 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     7021 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     7081 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     7141 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     7201 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     7261 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     7321 cgttgttgcc attgctgcag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     7381 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     7441 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     7501 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     7561 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     7621 ctcttgcccg gcgtcaacac gggataatac cgcgccacat agcagaactt taaaagtgct
     7681 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     7741 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     7801 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     7861 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     7921 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     7981 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     8041 attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat tcataccaga
     8101 tcaccgaaaa ctgtcctcca aatgtgtccc cctcacactc ccaaattcgc gggcttctgc
     8161 ctcttagacc actctaccct attccccaca ctcaccggag ccaaagccgc ggcccttccg
     8221 tttctttgct
//
