LOCUS       sequence-200248-        6772 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Tet-inducible expression of human CHMP4B 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 6772)
  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 6772)
  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_CHMP4B_LAP
FEATURES             Location/Qualifiers
     source          1..6772
                     /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(1732..1756)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1776..1796
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1809..1853
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1893..1916
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1944..1946,1947..1949,1950..2663)
                     /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(1989..2010)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2250..2269)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2724..2741)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2730..2954
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3087..3106)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3238..3285
                     /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             3293..4315
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3293..4315
                     /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    4445..4566
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4482..4501)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4536..4555
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4615..4631)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4615..4631)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4628..4650)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4639..4655
                     /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(4663..4669,4670..4687,4688..4693))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4708..4729
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4846..4863)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5017..5605)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5097..5116)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5776..6567,6568..6636))
                     /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     6399..6418
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6637..6741)
                     /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 gtcggtgttc gggaagctgt
     1081 tcggggctgg agggggtaag gccggcaagg gcggcccgac cccccaggag gccatccagc
     1141 ggctgcggga cacggaagag atgttaagca agaaacagga gttcctggag aagaaaatcg
     1201 agcaggagct gacggccgcc aagaagcacg gcaccaaaaa caagcgcgcg gccctccagg
     1261 cactgaagcg taagaagagg tatgagaagc agctggcgca gatcgacggc acattatcaa
     1321 ccatcgagtt ccagcgggag gccctggaga atgccaacac caacaccgag gtgctcaaga
     1381 acatgggcta tgccgccaag gccatgaagg cggcccatga caacatggac atcgataaag
     1441 ttgatgagtt aatgcaggac attgctgacc agcaagaact tgcagaggag atttcaacag
     1501 caatttcgaa acctgtaggg tttggagaag agtttgacga ggatgagctc atggcggaat
     1561 tagaagaact agaacaggag gaactagaca agaatttgct ggaaatcagt ggacccgaaa
     1621 cagtccctct accaaatgtt ccctctatag ccctaccatc aaaacccgcc aagaagaaag
     1681 aagaggagga cgacgacatg aaggaattgg agaactgggc tggatccatg tacccaactt
     1741 tcttgtacaa agtggtaccg gtcgccaccg caagcgagaa tctttatttt cagggcgccg
     1801 ccaaattcaa agaaaccgct gctgctaaat tcgaacgcca gcacatggac agcggaggtg
     1861 gaggttcgag cggtccctcg ggttcgtcga gcctggaagt tctgttccag gggcccctgt
     1921 cgtcgagcgg tccctcgggt tcgatggtga gcaaaggcga ggagctgttc accggggtgg
     1981 tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtgtccggcg
     2041 agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc accaccggca
     2101 agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg cagtgcttca
     2161 gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg cccgaaggct
     2221 acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg
     2281 tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg
     2341 aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac aacgtctata
     2401 tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc cacaacatcg
     2461 aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc
     2521 ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc aaagacccca
     2581 acgagaagcg cgatcacatg gtccttaagg agttcgtgac cgccgccggg atcactctcg
     2641 gcatggacga gctgtacaag taagatatcc agcacagtgg cggccgctcg agtctagagg
     2701 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     2761 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2821 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2881 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2941 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3001 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3061 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3121 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3181 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa
     3241 gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc
     3301 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     3361 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     3421 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     3481 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     3541 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     3601 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     3661 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     3721 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     3781 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     3841 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     3901 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     3961 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     4021 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     4081 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     4141 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     4201 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     4261 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt
     4321 actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4381 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     4441 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4501 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4561 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     4621 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4681 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4741 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     4801 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4861 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4921 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     4981 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5041 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5101 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5161 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5221 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5281 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5341 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5401 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5461 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5521 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5581 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5641 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5701 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5761 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5821 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5881 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     5941 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6001 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6061 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6121 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6181 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6241 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6301 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6361 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6421 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6481 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6541 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6601 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6661 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6721 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tc
//
