LOCUS       sequence-200235-        6739 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Tet-inducible expression of human CHMP2B 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 6739)
  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 6739)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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_CHMP2B_LAP
FEATURES             Location/Qualifiers
     source          1..6739
                     /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(1699..1723)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1743..1763
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1776..1820
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             1860..1883
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(1911..1913,1914..1916,1917..2630)
                     /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(1956..1977)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2217..2236)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(2691..2708)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2697..2921
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3054..3073)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3205..3252
                     /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             3260..4282
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3260..4282
                     /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    4412..4533
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4449..4468)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4503..4522
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4582..4598)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4582..4598)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4595..4617)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4606..4622
                     /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(4630..4636,4637..4654,4655..4660))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4675..4696
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4813..4830)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4984..5572)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5064..5083)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5743..6534,6535..6603))
                     /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     6366..6385
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6604..6708)
                     /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 ggcgtccctc ttcaagaaga
     1081 aaaccgtgga tgatgtaata aaggaacaga atcgagagtt acgaggtaca cagagggcta
     1141 taatcagaga tcgagcagct ttagagaaac aagaaaaaca gctggaatta gaaattaaga
     1201 aaatggccaa gattggtaat aaggaagctt gcaaagtttt agccaaacaa cttgtgcatc
     1261 tacggaaaca gaagacgaga acttttgctg taagttcaaa agttacttct atgtctacac
     1321 aaacaaaagt gatgaattcc caaatgaaga tggctggagc aatgtctacc acagcaaaaa
     1381 caatgcaggc agttaacaag aagatggatc cacaaaagac attacaaaca atgcagaatt
     1441 tccagaagga aaacatgaaa atggaaatga ctgaagaaat gatcaatgat acacttgatg
     1501 acatctttga cggttctgat gacgaagaag aaagccagga tattgtgaat caagttcttg
     1561 atgaaattgg aattgaaatt tctggaaaga tggccaaagc tccatcagct gctcgaagct
     1621 taccatctgc ctctacttca aaggctacaa tctcagatga agagattgaa cggcaactca
     1681 aggctttagg agtagattac ccaactttct tgtacaaagt ggtaccggtc gccaccgcaa
     1741 gcgagaatct ttattttcag ggcgccgcca aattcaaaga aaccgctgct gctaaattcg
     1801 aacgccagca catggacagc ggaggtggag gttcgagcgg tccctcgggt tcgtcgagcc
     1861 tggaagttct gttccagggg cccctgtcgt cgagcggtcc ctcgggttcg atggtgagca
     1921 aaggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1981 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     2041 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     2101 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     2161 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     2221 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     2281 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     2341 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     2401 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     2461 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     2521 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc cttaaggagt
     2581 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa gatatccagc
     2641 acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     2701 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2761 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2821 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2881 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     2941 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     3001 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3061 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3121 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3181 ttagggtgat ggttcacgta cctagaagtt cctattccga agttcctatt ctctagaaag
     3241 tataggaact tccttggcca aaaagcctga actcaccgcg acgtctgtcg agaagtttct
     3301 gatcgaaaag ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg
     3361 tgctttcagc ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga
     3421 tggtttctac aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc
     3481 ggaagtgctt gacattgggg aattcagcga gagcctgacc tattgcatct cccgccgtgc
     3541 acagggtgtc acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt
     3601 cgcggaggcc atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc
     3661 attcggaccg caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc
     3721 tgatccccat gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc
     3781 gcaggctctc gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt
     3841 gcacgcggat ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat
     3901 tgactggagc gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg
     3961 gaggccgtgg ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga
     4021 gcttgcagga tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta
     4081 tcagagcttg gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc
     4141 aatcgtccga tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc
     4201 cgtctggacc gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac
     4261 tcgtccgagg gcaaaggaat agcacgtact acgagatttc gattccaccg ccgccttcta
     4321 tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg
     4381 ggatctcatg ctggagttct tcgcccaccc caacttgttt attgcagctt ataatggtta
     4441 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4501 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgtataccgt cgacctctag
     4561 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4621 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4681 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4741 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     4801 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     4861 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     4921 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     4981 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5041 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5101 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5161 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5221 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5281 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5341 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5401 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     5461 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5521 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5581 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     5641 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     5701 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     5761 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     5821 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     5881 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     5941 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6001 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6061 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6121 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6181 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6241 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6301 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6361 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6421 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6481 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6541 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6601 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     6661 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     6721 aaaagtgcca cctgacgtc
//
