LOCUS       sequence-200249-        6766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet-inducible expression of human CHMP3 with a C-terminal GFP and 
            long linker (LAP tag), compatible with Flp-In recombination for 
            stable integration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6766)
  AUTHORS   Mierzwa BE, Chiaruttini N, Redondo-Morata L, von Filseck JM, Konig 
            J, Larios J, Poser I, Muller-Reichert T, Scheuring S, Roux A, 
            Gerlich DW
  TITLE     Dynamic subunit turnover in ESCRT-III assemblies is regulated by 
            Vps4 to mediate membrane remodelling during cytokinesis.
  JOURNAL   Nat Cell Biol. 2017 Jul;19(7):787-798. doi: 10.1038/ncb3559. Epub 
            2017 Jun 12.
  PUBMED    28604678
REFERENCE   2  (bases 1 to 6766)
  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: "10.1038/ncb3559"; 
            journalName: "Nat Cell Biol"; date: "2017-07"; volume: "19"; issue: 
            "7"; pages: "787-798"
COMMENT     Sequence Label: pTO_CHMP3_LAP
FEATURES             Location/Qualifiers
     source          1..6766
                     /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"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    1035..1059
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1726..1750)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1770..1790
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1803..1847
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1887..1910
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1938..1940,1941..1943,1944..2657)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1983..2004)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2244..2263)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2718..2735)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2724..2948
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3081..3100)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3232..3279
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3287..4309
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3287..4309
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4439..4560
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4476..4495)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4530..4549
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4609..4625)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4609..4625)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4622..4644)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4633..4649
                     /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(4657..4663,4664..4681,4682..4687))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4702..4723
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4840..4857)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5011..5599)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5091..5110)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5770..6561,6562..6630))
                     /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     6393..6412
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6631..6735)
                     /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 ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttggtaccg agctcggatc cactagtcca gtgtggtgga
     1021 attctgcaga tatcacaagt ttgtacaaaa aagttggcat ggggctgttt ggaaagaccc
     1081 aggagaagcc gcccaaagaa ctggtcaatg agtggtcatt gaagataaga aaggaaatga
     1141 gagttgttga caggcaaata agggatatcc aaagagaaga agaaaaagtg aaacgatctg
     1201 tgaaagatgc tgccaagaag ggccagaagg atgtctgcat agttctggcc aaggagatga
     1261 tcaggtcaag gaaggctgtg agcaagctgt atgcatccaa agcacacatg aactcagtgc
     1321 tcatggggat gaagaaccag ctcgcggtct tgcgagtggc tggttccctg cagaagagca
     1381 cagaagtgat gaaggccatg caaagtcttg tgaagattcc agagattcag gccaccatga
     1441 gggagttgtc caaagaaatg atgaaggctg ggatcataga ggagatgtta gaggacactt
     1501 ttgaaagcat ggacgatcag gaagaaatgg aggaagaagc agaaatggaa attgacagaa
     1561 ttctctttga aattacagca ggggccttgg gcaaagcacc cagtaaagtg actgatgccc
     1621 ttccagagcc agaacctcca ggagcgatgg ctgcctcaga ggatgaggag gaggaggaag
     1681 aggctctgga ggccatgcag tcccggctgg ccacactccg cagctaccca actttcttgt
     1741 acaaagtggt accggtcgcc accgcaagcg agaatcttta ttttcagggc gccgccaaat
     1801 tcaaagaaac cgctgctgct aaattcgaac gccagcacat ggacagcgga ggtggaggtt
     1861 cgagcggtcc ctcgggttcg tcgagcctgg aagttctgtt ccaggggccc ctgtcgtcga
     1921 gcggtccctc gggttcgatg gtgagcaaag gcgaggagct gttcaccggg gtggtgccca
     1981 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     2041 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     2101 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     2161 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     2221 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     2281 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     2341 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     2401 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     2461 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     2521 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     2581 agcgcgatca catggtcctt aaggagttcg tgaccgccgc cgggatcact ctcggcatgg
     2641 acgagctgta caagtaagat atccagcaca gtggcggccg ctcgagtcta gagggcccgt
     2701 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2761 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2821 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     2881 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     2941 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3001 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3061 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3121 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3181 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct
     3241 attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact
     3301 caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat
     3361 gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata
     3421 tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca
     3481 ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag
     3541 cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac
     3601 cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga
     3661 tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac
     3721 atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat
     3781 ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga
     3841 ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac
     3901 ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca
     3961 atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac
     4021 gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat
     4081 gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc
     4141 agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg
     4201 tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc
     4261 cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg
     4321 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
     4381 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     4441 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     4501 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4561 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     4621 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4681 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4741 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4801 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4861 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4921 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4981 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5041 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5101 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5161 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5221 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5281 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5341 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5401 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5461 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5521 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5581 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5641 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5701 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5761 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5821 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5881 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5941 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6001 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6061 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6121 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6181 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6241 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6301 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6361 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6421 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6481 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6541 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6601 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6661 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6721 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
