LOCUS       sequence-220534-        9504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Muscle specific zebrafish expression of mKate2 fused to dominant 
            negative CDC42.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9504)
  AUTHORS   Hall TE, Martel N, Ariotti N, Xiong Z, Lo HP, Ferguson C, Rae J, Lim
            YW, Parton RG
  TITLE     In vivo cell biological screening identifies an endocytic capture 
            mechanism for T-tubule formation.
  JOURNAL   Nat Commun. 2020 Jul 24;11(1):3711. doi: 10.1038/s41467-020-17486-w.
  PUBMED    32709891
REFERENCE   2  (bases 1 to 9504)
  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: "Nat 
            Commun."; date: "2020-07-24"; volume: "11(1):3711. doi"; pages: " 
            10.1038/s41467-020-17486-w"
COMMENT     Sequence Label: actc1b-mKate2-CDC42-DN
FEATURES             Location/Qualifiers
     source          1..9504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      26..35
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             32..727
                     /codon_start=1
                     /product="monomeric far-red fluorescent protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK
                     AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT
                     ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL
                     KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY
                     CDLPSKLGHR"
     protein_bind    complement(729..753)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1328..1348)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(1356..1372)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1388..1522
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1412..1431)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1466..1485
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(1961..2148,2149..2160)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(2194..2210)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2194..2210)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2247..2267)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2247..2265)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2286..2302)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2299..2321)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2310..2326
                     /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(2334..2340,2341..2358,2359..2364))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2379..2400
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2517..2534)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2688..3276)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2768..2787)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3447..4238,4239..4307))
                     /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     4070..4089
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4308..4412)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4575..4596)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(4945..4956,4957..5119)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     5492..5508
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5528..5548
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
ORIGIN
        1 gcaagtttgt acaaaaaagc aggctgccac catggtgagc gagctgatta aggagaacat
       61 gcacatgaag ctgtacatgg agggcaccgt gaacaaccac cacttcaagt gcacatccga
      121 gggcgaaggc aagccctacg agggcaccca gaccatgaga atcaaggcgg tcgagggcgg
      181 ccctctcccc ttcgccttcg acatcctggc taccagcttc atgtacggca gcaaaacctt
      241 catcaaccac acccagggca tccccgactt ctttaagcag tccttccccg agggcttcac
      301 atgggagaga gtcaccacat acgaagacgg gggcgtgctg accgctaccc aggacaccag
      361 cctccaggac ggctgcctca tctacaacgt caagatcaga ggggtgaact tcccatccaa
      421 cggccctgtg atgcagaaga aaacactcgg ctgggaggcc tccaccgaga ccctgtaccc
      481 cgctgacggc ggcctggaag gcagagccga catggccctg aagctcgtgg gcgggggcca
      541 cctgatctgc aacttgaaga ccacatacag atccaagaaa cccgctaaga acctcaagat
      601 gcccggcgtc tactatgtgg acagaagact ggaaagaatc aaggaggccg acaaagagac
      661 ctacgtcgag cagcacgagg tggctgtggc cagatactgc gacctcccta gcaaactggg
      721 gcacagagac ccagctttct tgtacaaagt ggctcagacg atcaagtgcg tcgttgttgg
      781 tgatggtgca gtgggtaaaa actgtctatt aatctcctat acaactaata aattcccctc
      841 cgaatatgta ccaacggtct ttgataacta tgcagttacg gttatgattg gtggtgagcc
      901 gtacaccctg gggctgtttg atactgcagg tcaggaagat tacgatagat tacgacccct
      961 gagttaccct cagacagatg tcttcttagt ctgtttctca gttgtttcgc cttcttcatt
     1021 tgaaaacgtc aaagaaaagt gggtacccga gatcacccac cactgtccaa agaccccttt
     1081 cctgctggtg gggacgcaga ttgatctgag agatgatcct tctactatcg aaaagcttgc
     1141 caagaacaaa cagaaaccca tcactccaga gacggcagag aagctggccc gcgatctgaa
     1201 ggctgtcaaa tacgtggaat gctccgttct gacgcagaaa ggcctaaaga acgtatttga
     1261 tgaggcgatt ttggcagccc tggagccccc agagcccaag aagaagcgta aatgtgtgct
     1321 gctatgacaa ctttattata catagttgat aattcactgg ccgtcgtttt acggtaccat
     1381 cgatgatgat ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     1441 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     1501 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     1561 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga
     1621 ttatgatcct ctagatcaga tctgcgaaga tacggccacg ggtgctcttg atcctgtggc
     1681 tgattttgga ctgtgctgct cgcagctgct gatgaatcac atacttcctc cattttcttc
     1741 cactgattga ctgttataat ttccctaatt tccaggtcaa ggtgctgtgc attgtggtaa
     1801 tagatgtgac atgacgtcac ttccaaagga ccaatgaaca tgtctgacca atttcatata
     1861 atgtgaaaac gattttcata ggcagaataa ataacattta aattaaactg ggcatcagcg
     1921 caattcaatt ggtttggtaa tagcaaggga aaatagaatg aagtgatctc caaaaaataa
     1981 gtactttttg actgtaaata aaattgtaag gagtaaaaag tacttttttt tctaaaaaaa
     2041 tgtaattaag taaaagtaaa agtattgatt tttaattgta ctcaagtaaa gtaaaaatcc
     2101 ccaaaaataa tacttaagta cagtaatcaa gtaaaattac tcaagtactt tacacctctg
     2161 gttcttgacc ccctaccttc agcaagccca gcagatccac tagttctaga gcggccgcca
     2221 ccgcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg cttggcgtaa
     2281 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     2341 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     2401 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     2461 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     2521 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     2581 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2641 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2701 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2761 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2821 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2881 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2941 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     3001 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     3061 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3121 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3181 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3241 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3301 gggtctgacg ctcagtggaa cgaaaactca cgttaaggca ttttggtcat gagattatca
     3361 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3421 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3481 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3541 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     3601 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3661 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3721 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3781 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3841 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3901 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3961 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     4021 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     4081 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4141 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4201 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4261 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4321 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4381 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     4441 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     4501 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     4561 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     4621 tcaaagggcg aaaaaccgtc tatcagggcg atggcccacg ctggctaaga actcatcagc
     4681 ctccccggtc catctaccca cgtaccaatg caccaattgg ccacaatgac ggctactaca
     4741 tggtgccatt ccttcctctt tataggaatg gagactacct cctgtccaac aatgctcttg
     4801 gatacgagta cgcctacctg ttggacccag gtcattgcac aacaccagaa atgccctctg
     4861 atctgcaaaa gacgtgaata tctgttcaga cacccatatc cactctgttc cacacaggtc
     4921 agaggtttgt ccaggagttc ttgacagagg tgtaaaaagt actcaaaaat tttactcaag
     4981 tgaaagtaca agtacttagg gaaaatttta ctcaattaaa agtaaaagta tctggctaga
     5041 atcttacttg agtaaaagta aaaaagtact ccattaaaat tgtacttgag tattaaggaa
     5101 gtaaaagtaa aagcaagaaa gaaaactaga gattcttgtt taagctttta atctcaaaaa
     5161 acattaaatg aaatgcatac aaggttttat cctgctttag aactgtttgt atttaattat
     5221 caaactataa gacagacaat ctaatgccag tacacgctac tcaaagttgt aaaacctcag
     5281 atttaacttc agtagaagct gattctcaaa attgttagtg tcaagcctag ctcttttggg
     5341 gctgaaaagc aatcctgcag tgctgaaaag cctctcacag gcagccgatg cgggaagagg
     5401 tgtattagtc ttgatagaga ggctgcaaat agcaggaaac gtgagcagag actccctggt
     5461 gtctgaaaca caggccagat gggccctcga gcaggaaaca gctatgacca tgattacgcc
     5521 aagctatcaa ctttgtatag aaaagttggc acgcgcagat tccgtgtggg cctggtaata
     5581 tcttaccatt ttaagctatt ttaagattag ggaaaaaaat taaataaaac tactttaaaa
     5641 ttactttcct taaaaagcaa catatttatt tagtttttgt ttaactggct caagattgta
     5701 atattttact tttacatgtt tttttaagta cctaaatgtg ttcaaaagta aagtatgtta
     5761 ttgttttttt tttttaatgt tagctaagct ttaaattgta atatattact tttaaaagtg
     5821 actttttaaa aatgtaaata aaaaagtaac tataaatgtg taatgcatgt tatttctgtg
     5881 ggtttttaat tttcatctca actgggctta caattttaac ataataaaaa tcaaatcaaa
     5941 tcaaatcagg cccttatttg tcacatacac tcttgtatac tgaaataaac taaatgttga
     6001 gaaaaattta aatacaccaa aataacgcat accttttttg taattaccta agtaaaacat
     6061 ttagttcctt ttttaagtga ctcaagattg taatctatta attgtaaaag ataaaactag
     6121 tgatcaatct gacgtgcttc tatgcagcct gtgagattaa ttgtgatgtt taattattaa
     6181 acaatttaaa tggatcaaac atttcacatg tgcttattgt gtccaacaaa accagcagaa
     6241 gcacttaaaa atattgcatg acaacatgag tttttaatgt aaattatata tacttcctaa
     6301 acgaagttaa tcatatttct aaaataataa tgtcattgaa atatttaaat aaatgataag
     6361 ctttagttga ttcaacataa aattgaaagc aataaggact tttttttttt acagcggtga
     6421 agcaaaagca tgtaataatc atataatata ataaataaat aaataaataa tggctcttgg
     6481 tgcaaatatg aacactttaa ataaatatag aataaatatg atcaataagt cataacgatg
     6541 tgttttatgt tgattataat ttaaacatgt ttctaacgca agttttaaaa actgttttaa
     6601 agtattattt taagttataa gatcaatttg actcaacatt ttgtagtgaa aatatgttgt
     6661 gtaaaattaa tataatatac actataaaaa tgcagggttc cacacaatac attaatgttg
     6721 tcccaacaca aatcgattaa gttaacaaat atatgtggat tgaacataaa aaattaagtt
     6781 gtcccaatga aatctcaaga attgtgttgt ttcagctcat tttaaataag tagttttaac
     6841 aagtaaaaaa taacatattt ttgagtgtgt atatatatat attacaaaat ataataatat
     6901 aaaatgtaaa agatataaac catgctacat atatttaaga gctattttgc acattacttt
     6961 tttgcaagcc attttagtgt tttttttaca aatatatatt agctaaatat acaatatctt
     7021 aaatatacaa ttaatcagac atgcaattaa tgcatttaac ctacataaaa atacatgatc
     7081 aacatatgtt ttatgctgat tacaactaaa tcaaacgtaa tgaaattaat ttataaaagc
     7141 gatttaaata tgtcaatatt ttaagttaaa atcacttgaa acctactata aatgaactta
     7201 aattaaaaaa atctccttta attccaatgc aacagtgttg ttttttcagc tgaggacccc
     7261 cggtgcagat ggttggcccc cggcccgcta ttctaaggaa gcctttctta attttagggc
     7321 ggcgctgcat gcgctcagat cagattcacg ggtggagagg gttattttct cttttttatt
     7381 ttttcttgcc tgctcatatt tgggcctgtg tgaaccccag gctccgcctc ggcccccagg
     7441 agccgctctt atttaaaggg gatcctcgcc ttcagtcttc gtcccagtgg aagcatcagt
     7501 cttgtgataa caacttggtg agtcctacag accagcgctc gactctaggc tttaaatgca
     7561 ttgatttctc tggattagct accgggagag ttttaacttt gcttattagt cttgtgctcg
     7621 aactgctagg tttaaagagt gttttctcta atgcatgcat gaagcgacag ctgaaaacca
     7681 gaaatagagc gtttgtcctc ctgttaaact ctagcctttc tttggcattt gtcagtccga
     7741 gtcttcagct gccggcagga gattgggttt cagtaacact tgctgagcac aaactacacg
     7801 tgctttcttc agatttaatt cattattaat cattctagat ttaaatgatg ttcttactca
     7861 ttgctgatta ttttagcctt gcggagcaga ttaaatgagc acgtgtttca ttaagattga
     7921 tttaaatata attaatattt caccactgta gttttaaaat tgccttctaa gtcatggttg
     7981 atttttaata ggtatcggct ctaattagat tactattttg gtttttaaat gttatttcta
     8041 tttcatttgt aggctcgttt ttaatgtgat tttattagat tctagtttta aaatcatctc
     8101 ttagccttaa tgagcagact gaatgaacat gtttcattaa gattaacttc attataatga
     8161 attatttaat cattctagtg ggttataaat cagctctatt taaattaatt gtcgtgttta
     8221 tttctattta atttttgatt attttttcag ggtcgttttt atgtgatttt ttcccgtttc
     8281 taattttaaa ctaattgttt gatttttata tgattcgact ctttttaaat gaattgtttt
     8341 gcttttttta ttgagcatat atatattata caatttcaat caacaattgt tgtgcttttt
     8401 aaatgttgtt tctatttcat ttctagggtc gattttattc gattctagtt ttaaaatcgt
     8461 gatttttaac ttctaacctt actgagcaga ctgaatgaac atgtttcatc aagattaact
     8521 tcattatagt taattattga atcgtgctag tggtttataa atcagctcta tttaaattaa
     8581 tcatcatcat tatttctatt taatttttat tatttcttta ggattatttt tatgtgattt
     8641 ttttcacgtt ctagttttaa actcaatgct cattactgct cacccttgct gagcagtctt
     8701 tcgttaagat taaattaatt aataaccatt ctagttttaa aacagtgtct taaggcatag
     8761 ctgatatttt acaagtttac tatttaatat atctcctttc tttttttagg gtcgatttca
     8821 tctgatttga ttaattttta ttatcttcac taagcttttt gttctaggtt ttattctcag
     8881 tattattact gttatatttg aactgaattt ctcctcatag tttattcatt ttatttattt
     8941 attttttgtt tattcgctat tttgtatttt agtttttaat tagaaatttt attagaatcg
     9001 ttttgaacta gtttttatgc actctaacat taatattaaa tcataactat tatttttatt
     9061 ataatttatt tgtgtttatc tttttgtgta gctttcatgt gtttttattt tagttttatt
     9121 atgtttgcat tgaatttttt ggactagttt atataacgat atacagaaaa gccatgacaa
     9181 caaatgtttt tataatacct attaagtata agctaatcag atttccacta ttctgttcaa
     9241 agttaacatt attagccatt ctgatggatc atttgacatt tgtcatatgt ctttatgaac
     9301 actgctcttt cagatgccac ttttaaagct gttttttaca gtaaatgaac gtttattatt
     9361 atgacattat ttcctaaggc ctctagagta cagcaggatg tggcgtgtgc attaccgtgt
     9421 aattatgctg actgtaaatg tgcatttata gactgtcagc atgtctgact gtgcgctgtt
     9481 gtgattggtc ctgcacagac caag
//
