LOCUS       sequence-443365-        7615 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 the NS3 cleavage 
            site, along with a blue fluorescent protein..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7615)
  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 7615)
  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-NS3-Blue
FEATURES             Location/Qualifiers
     source          1..7615
                     /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"
     misc_feature    2365..2430
                     /label=MCS
                     /note="multiple cloning site"
     regulatory      2438..2447
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2444..2446,2447..2449,2450..3163)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2489..2510)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2750..2769)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3097..3118
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3206..3223)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3212..3436
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3482..3910
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3569..3588)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3779..3800
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3919..3939)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3924..4253
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4104..4239
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4166..4185
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4320..5114
                     /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(4374..4393)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4984..5003
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5288..5409
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5325..5344)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5379..5398
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5458..5474)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5458..5474)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5471..5493)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5482..5498
                     /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(5506..5512,5513..5530,5531..5536))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5551..5572
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5689..5706)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5860..6448)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5940..5959)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6619..7410,7411..7479))
                     /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     7242..7261
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7480..7584)
                     /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 gtttgatgag atggaagagt 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 gcgctccgga ctcagatctc gagctcaagc ttcgaattct
     2401 gcagtcgacg gtaccgcggg cccgggatcc accggtcgcc accatggtga gcaagggcga
     2461 ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca
     2521 caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa
     2581 gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgtc
     2641 ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa
     2701 gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa
     2761 ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct
     2821 gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta
     2881 ctttagcgac aacgtctata tcaccgccga caagcagaag aacggcatca aggccaactt
     2941 caagatccgc cacaacatcg aggacggcgg cgtgcagctc gccgaccact accagcagaa
     3001 cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc
     3061 caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac
     3121 cgccgccggg atcactctcg gcatggacga gctgtacaag taagcggccg cgactctaga
     3181 gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     3241 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     3301 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     3361 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     3421 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     3481 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     3541 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     3601 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     3661 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     3721 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     3781 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     3841 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     3901 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     3961 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     4021 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4081 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     4141 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     4201 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     4261 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     4321 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     4381 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     4441 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     4501 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     4561 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     4621 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     4681 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     4741 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     4801 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     4861 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     4921 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     4981 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     5041 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     5101 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     5161 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     5221 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     5281 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     5341 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     5401 tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc
     5461 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     5521 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     5581 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     5641 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5701 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5761 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5821 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5881 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5941 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     6001 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     6061 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     6121 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     6181 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     6241 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6301 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6361 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6421 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6481 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6541 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6601 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6661 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6721 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6781 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6841 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6901 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6961 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     7021 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     7081 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     7141 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     7201 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     7261 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7321 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7381 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7441 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7501 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7561 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
