LOCUS       sequence-443350-        7548 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of CHMP2A fused to the hepatitis C virus 
            protease NS3 through a short linker containing a mutated NS3 
            cleavage site, along with a green fluorescent protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7548)
  AUTHORS   Wang H, Gallet B, Moriscot C, Pezet M, Chatellard C, Kleman JP, 
            Gottlinger H, Weissenhorn W, Boscheron C
  TITLE     An Inducible ESCRT-III Inhibition Tool to Control HIV-1 Budding.
  JOURNAL   Viruses. 2023 Nov 22;15(12):2289. doi: 10.3390/v15122289.
  PUBMED    38140530
REFERENCE   2  (bases 1 to 7548)
  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: "Viruses."; 
            date: "2023-11-22"; volume: "15(12):2289. doi"; pages: " 
            10.3390/v15122289"
COMMENT     Sequence Label: pCHMP2A-mut-NS3-Green
FEATURES             Location/Qualifiers
     source          1..7548
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1601..1624
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2315..2338
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2360..3064
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGMDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELY"
     regulatory      2615..2624
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3139..3156)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3145..3369
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3415..3843
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3502..3521)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3712..3733
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3852..3872)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3857..4186
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4037..4172
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4099..4118
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4253..5047
                     /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(4307..4326)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4917..4936
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5221..5342
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5258..5277)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5312..5331
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5391..5407)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5391..5407)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5404..5426)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5415..5431
                     /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(5439..5445,5446..5463,5464..5469))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5484..5505
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5622..5639)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5793..6381)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5873..5892)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6552..7343,7344..7412))
                     /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     7175..7194
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7413..7517)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttatgg acctgctgtt cggcagaaga aagacccctg aggaactgct
      961 gcggcagaac cagagagccc tgaacagagc catgagagag ctggacagag agcggcagaa
     1021 gctggaaacc caagagaaga agatcattgc cgacatcaag aagatggcca agcagggcca
     1081 gatggacgcc gtcagaatca tggccaaaga cctcgtgcgg actcggagat acgtgcggaa
     1141 gttcgtgctg atgcgggcca atatccaggc cgtgtctctg aagatccaga cactgaagtc
     1201 caacaacagc atggcccagg ccatgaaggg cgtgacaaag gccatgggca ccatgaacag
     1261 acagctgaag ctgccccaga tccagaaaat catgatggaa ttcgagcggc aggccgagat
     1321 catggacatg aaggaagaga tgatgaacga cgccatcgac gatgccatgg gcgacgaaga
     1381 ggatgaagag gaatccgacg ccgtggtgtc ccaggtgctg gatgaactgg gactgagcct
     1441 gaccgacgag ctgagcaatc tgcctagcac aggcggatct ctgtctgtgg ctgccggcgg
     1501 aaagaaagcc gaagctgctg cttctgccct ggccgatgct gatgccgatc tggaagagag
     1561 actgaagaac ctgcggcggg atggcggctc tacaggcaat tggagccatc ctcagttcga
     1621 gaaaggcagc ggcagctgtc aagagtccgg aagcatggga tctggcagcc agcatctgcc
     1681 tggcgctgga tcttctggcg gatctcacat ggccagcatg aagaaaaagg gcagcgtggt
     1741 catcgtgggc agaatcaacc tgagcggcga taccgcctac gctcagcaga caagaggcga
     1801 ggaaggctgc caagagacaa gccagaccgg cagagacaag aaccaggtgg aaggcgaggt
     1861 gcagatcgtg tctacagcta cccagacctt cctggccacc agcatcaatg gcgtgctgtg
     1921 gacagtgtat cacggcgctg gcaccaggac aatcgcctct ccaaaaggcc ccgtgacaca
     1981 gatgtacacc aatgtggaca aagatctggt gggttggcag gcaccgcaag gcagccgtag
     2041 cctgacgccg tgcacctgtg gcagttccga tctgtatctg gttacccgcc atgcggacgt
     2101 gattccggtt cgtcgccgtg gcgatagtcg tggttccctg ctgtcaccgc gcccgatctc
     2161 gtacctgaaa ggttcatcgg gtggtccgct gctgtgcccg gcaggtcacg cagttggtat
     2221 ttttcgtgca gcagtcagca cccgcggcgt ggctaaagcg gttgatttca tcccggtgga
     2281 atccctggaa accacgatgc gttctggcag cggcgactac aaagacgatg atgataaatc
     2341 ctccggctcc ggcagcgcta tggtgagcaa gggcgaggag gataacatgg cctctctccc
     2401 agcgacacat gagttacaca tctttggctc catcaacggt atggactttg acatggtggg
     2461 tcagggcacc ggcaatccaa atgatggtta tgaggagtta aacctgaagt ccaccaaggg
     2521 tgacctccag ttctccccct ggattctggt ccctcatatc gggtatggct tccatcagta
     2581 cctgccctac cctgacggga tgtcgccttt ccaggccgcc atggtagatg gctccggata
     2641 ccaagtccat cgcacaatgc agtttgaaga tggtgcctcc cttactgtta actaccgcta
     2701 cacctacgag ggaagccaca tcaaaggaga ggcccaggtg aaggggactg gtttccctgc
     2761 tgacggtcct gtgatgacca actcgctgac cgctgcggac tggtgcaggt cgaagaagac
     2821 ttaccccaac gacaaaacca tcatcagtac ctttaagtgg agttacacca ctggaaatgg
     2881 caagcgctac cggagcactg cgcggaccac ctacaccttt gccaagccaa tggcggctaa
     2941 ctatctgaag aaccagccga tgtacgtgtt ccgtaagacg gagctcaagc actccaagac
     3001 cgagctcaac ttcaaggagt ggcaaaaggc ctttaccgat gtgatgggca tggacgagct
     3061 gtacgtagga attctgcaga tatccagcac agtggcggcc gctcgagtct agagggcccg
     3121 tttaaacccg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     3181 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     3241 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     3301 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     3361 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     3421 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     3481 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     3541 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     3601 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3661 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3721 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3781 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3841 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg
     3901 cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg
     3961 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc
     4021 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     4081 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt
     4141 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc
     4201 ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga
     4261 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     4321 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     4381 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     4441 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     4501 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     4561 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     4621 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     4681 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     4741 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     4801 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     4861 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     4921 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     4981 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     5041 cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca
     5101 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     5161 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     5221 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     5281 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     5341 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     5401 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     5461 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     5521 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5581 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5641 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5701 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5761 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5821 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5881 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5941 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     6001 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     6061 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     6121 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     6181 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     6241 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     6301 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     6361 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     6421 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     6481 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     6541 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     6601 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     6661 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     6721 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     6781 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     6841 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     6901 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     6961 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     7021 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     7081 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     7141 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     7201 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     7261 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     7321 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     7381 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     7441 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     7501 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
