LOCUS       sequence-443356-        7554 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of CHMP4B fused to the hepatitis C virus 
            protease NS3 through a short linker containing 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 7554)
  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 7554)
  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: pCHMP4B-mut-NS3-Green
FEATURES             Location/Qualifiers
     source          1..7554
                     /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             1607..1630
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             2321..2344
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             2366..3070
                     /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      2621..2630
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(3145..3162)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3151..3375
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3421..3849
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3508..3527)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3718..3739
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3858..3878)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3863..4192
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4043..4178
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4105..4124
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4259..5053
                     /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(4313..4332)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4923..4942
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5227..5348
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5264..5283)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5318..5337
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5397..5413)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5397..5413)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5410..5432)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5421..5437
                     /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(5445..5451,5452..5469,5470..5475))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5490..5511
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5628..5645)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5799..6387)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5879..5898)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6558..7349,7350..7418))
                     /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     7181..7200
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7419..7523)
                     /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 aagcttatga gcgtgttcgg caagctgttc ggagccggcg gaggaaaagc
      961 tggaaaaggc ggacctacac ctcaagaggc catccagaga ctgcgggaca ccgaggaaat
     1021 gctgagcaag aagcaagagt tcctcgagaa gaagatcgag caagagctga ccgccgccaa
     1081 gaagcacggc accaagaaca aaagagccgc tctgcaggcc ctgaagcgga agaagagata
     1141 cgagaagcag ctggcccaga tcgacggcac cctgagcaca atcgagttcc agagagaggc
     1201 cctggaaaac gccaacacca acaccgaggt gctgaagaac atgggctatg ccgccaaggc
     1261 catgaaggcc gctcacgaca acatggacat cgacaaggtg gacgagctga tgcaggatat
     1321 cgccgaccag caagaactgg ccgaagagat cagcaccgcc atctctaagc ctgtcggctt
     1381 cggcgaagag ttcgacgagg atgagctgat ggccgagctg gaagaactgg aacaagagga
     1441 actggataag aacctgctgg aaatcagcgg ccctgagaca gtgcccctgc ctaatgtgcc
     1501 ttctatcgcc ctgcctagca agcctgccaa gaagaaagag gaagaggacg acgacatgaa
     1561 ggaactcgag aactgggccg gctctatggg cggctctaca ggcaattgga gccatcctca
     1621 gttcgagaaa ggcagcggca gctgtcaaga gtccggaagc atgggatctg gcagccagca
     1681 tctgcctggc gctggatctt ctggcggatc tcacatggcc agcatgaaga aaaagggcag
     1741 cgtggtcatc gtgggcagaa tcaacctgag cggcgatacc gcctacgctc agcagacaag
     1801 aggcgaggaa ggctgccaag agacaagcca gaccggcaga gacaagaacc aggtggaagg
     1861 cgaggtgcag atcgtgtcta cagctaccca gaccttcctg gccaccagca tcaatggcgt
     1921 gctgtggaca gtgtatcacg gcgctggcac caggacaatc gcctctccaa aaggccccgt
     1981 gacacagatg tacaccaatg tggacaaaga tctggtgggt tggcaggcac cgcaaggcag
     2041 ccgtagcctg acgccgtgca cctgtggcag ttccgatctg tatctggtta cccgccatgc
     2101 ggacgtgatt ccggttcgtc gccgtggcga tagtcgtggt tccctgctgt caccgcgccc
     2161 gatctcgtac ctgaaaggtt catcgggtgg tccgctgctg tgcccggcag gtcacgcagt
     2221 tggtattttt cgtgcagcag tcagcacccg cggcgtggct aaagcggttg atttcatccc
     2281 ggtggaatcc ctggaaacca cgatgcgttc tggcagcggc gactacaaag acgatgatga
     2341 taaatcctcc ggctccggca gcgctatggt gagcaagggc gaggaggata acatggcctc
     2401 tctcccagcg acacatgagt tacacatctt tggctccatc aacggtatgg actttgacat
     2461 ggtgggtcag ggcaccggca atccaaatga tggttatgag gagttaaacc tgaagtccac
     2521 caagggtgac ctccagttct ccccctggat tctggtccct catatcgggt atggcttcca
     2581 tcagtacctg ccctaccctg acgggatgtc gcctttccag gccgccatgg tagatggctc
     2641 cggataccaa gtccatcgca caatgcagtt tgaagatggt gcctccctta ctgttaacta
     2701 ccgctacacc tacgagggaa gccacatcaa aggagaggcc caggtgaagg ggactggttt
     2761 ccctgctgac ggtcctgtga tgaccaactc gctgaccgct gcggactggt gcaggtcgaa
     2821 gaagacttac cccaacgaca aaaccatcat cagtaccttt aagtggagtt acaccactgg
     2881 aaatggcaag cgctaccgga gcactgcgcg gaccacctac acctttgcca agccaatggc
     2941 ggctaactat ctgaagaacc agccgatgta cgtgttccgt aagacggagc tcaagcactc
     3001 caagaccgag ctcaacttca aggagtggca aaaggccttt accgatgtga tgggcatgga
     3061 cgagctgtac gtaggaattc tgcagatatc cagcacagtg gcggccgctc gagtctagag
     3121 ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg
     3181 tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct
     3241 aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg
     3301 gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg
     3361 cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc
     3421 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3481 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3541 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3601 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc
     3661 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3721 gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct tttgatttat
     3781 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3841 acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     3901 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     3961 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     4021 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     4081 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct ctgcctctga
     4141 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctccc
     4201 gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat cgtttcgcat
     4261 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg
     4321 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc
     4381 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca
     4441 ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct
     4501 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga
     4561 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg
     4621 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat
     4681 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga
     4741 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg
     4801 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg
     4861 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat
     4921 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct
     4981 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga
     5041 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg
     5101 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt
     5161 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc
     5221 caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat
     5281 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat
     5341 gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca
     5401 tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     5461 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     5521 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     5581 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     5641 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5701 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5761 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5821 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5881 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5941 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     6001 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     6061 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     6121 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     6181 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     6241 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     6301 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     6361 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6421 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6481 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6541 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6601 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6661 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6721 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6781 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6841 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6901 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6961 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     7021 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     7081 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     7141 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     7201 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     7261 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     7321 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     7381 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7441 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7501 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
