LOCUS       sequence-220524-        9510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Muscle specific zebrafish expression of mKate2 fused to dominant 
            negative RhoAa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9510)
  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 9510)
  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-RhoAa-DN
FEATURES             Location/Qualifiers
     source          1..9510
                     /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(1334..1354)
                     /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(1362..1378)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1394..1528
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1418..1437)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1472..1491
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(1967..2154,2155..2166)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(2200..2216)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2200..2216)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2253..2273)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2253..2271)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2292..2308)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2305..2327)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2316..2332
                     /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(2340..2346,2347..2364,2365..2370))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2385..2406
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2523..2540)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2694..3282)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2774..2793)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3453..4244,4245..4313))
                     /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     4076..4095
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4314..4418)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4581..4602)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(4951..4962,4963..5125)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     5498..5514
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5534..5554
                     /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 ggtggctgcg attcgtaaga agcttgtaat
      781 cgttggagat gtagcctgtg gaaagacgtg tttactcatc gttttcagta aagaccagtt
      841 tcctgaggtt tacgttccca cagtgtttga gaactatgtc gctgatattg aggtggattc
      901 aaaacaggtg gaactggctc tatgggatac agctggccaa gaggactacg acaggctgag
      961 gcccctgtcc tacccagaca ctgatgtcat ccttatgtgc ttttccatag acagtcctga
     1021 cagtttggag aacatcccag agaaatggac gccagaagta aaacatttct gccctaatgt
     1081 tccaataata ctggtgggta ataaaaaaga tttacggaat gacgagcaca ctcgacggga
     1141 gctgcagaag atgaaacagg aaccagttaa accagaggag ggacgagaca tggctaatcg
     1201 catcaatgcc ttcggctacc ttgagtgttc agctaaaact aaggagggcg tgagggaagt
     1261 gtttgaaatg gccacaagag cggcgctcca agccaagaaa cgtggcaaga agaatgcctg
     1321 cgctttgcta tagcaacttt attatacata gttgataatt cactggccgt cgttttacgg
     1381 taccatcgat gatgatccag acatgataag atacattgat gagtttggac aaaccacaac
     1441 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     1501 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     1561 ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca aatgtggtat
     1621 ggctgattat gatcctctag atcagatctg cgaagatacg gccacgggtg ctcttgatcc
     1681 tgtggctgat tttggactgt gctgctcgca gctgctgatg aatcacatac ttcctccatt
     1741 ttcttccact gattgactgt tataatttcc ctaatttcca ggtcaaggtg ctgtgcattg
     1801 tggtaataga tgtgacatga cgtcacttcc aaaggaccaa tgaacatgtc tgaccaattt
     1861 catataatgt gaaaacgatt ttcataggca gaataaataa catttaaatt aaactgggca
     1921 tcagcgcaat tcaattggtt tggtaatagc aagggaaaat agaatgaagt gatctccaaa
     1981 aaataagtac tttttgactg taaataaaat tgtaaggagt aaaaagtact tttttttcta
     2041 aaaaaatgta attaagtaaa agtaaaagta ttgattttta attgtactca agtaaagtaa
     2101 aaatccccaa aaataatact taagtacagt aatcaagtaa aattactcaa gtactttaca
     2161 cctctggttc ttgaccccct accttcagca agcccagcag atccactagt tctagagcgg
     2221 ccgccaccgc ggtggagctc cagcttttgt tccctttagt gagggttaat tgcgcgcttg
     2281 gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac
     2341 aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc
     2401 acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg
     2461 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     2521 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     2581 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     2641 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     2701 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     2761 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     2821 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     2881 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     2941 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     3001 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     3061 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     3121 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     3181 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     3241 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     3301 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aaggcatttt ggtcatgaga
     3361 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     3421 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     3481 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     3541 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     3601 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     3661 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     3721 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg
     3781 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     3841 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     3901 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     3961 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     4021 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     4081 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     4141 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     4201 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     4261 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     4321 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     4381 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     4441 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     4501 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     4561 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     4621 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccacgctgg ctaagaactc
     4681 atcagcctcc ccggtccatc tacccacgta ccaatgcacc aattggccac aatgacggct
     4741 actacatggt gccattcctt cctctttata ggaatggaga ctacctcctg tccaacaatg
     4801 ctcttggata cgagtacgcc tacctgttgg acccaggtca ttgcacaaca ccagaaatgc
     4861 cctctgatct gcaaaagacg tgaatatctg ttcagacacc catatccact ctgttccaca
     4921 caggtcagag gtttgtccag gagttcttga cagaggtgta aaaagtactc aaaaatttta
     4981 ctcaagtgaa agtacaagta cttagggaaa attttactca attaaaagta aaagtatctg
     5041 gctagaatct tacttgagta aaagtaaaaa agtactccat taaaattgta cttgagtatt
     5101 aaggaagtaa aagtaaaagc aagaaagaaa actagagatt cttgtttaag cttttaatct
     5161 caaaaaacat taaatgaaat gcatacaagg ttttatcctg ctttagaact gtttgtattt
     5221 aattatcaaa ctataagaca gacaatctaa tgccagtaca cgctactcaa agttgtaaaa
     5281 cctcagattt aacttcagta gaagctgatt ctcaaaattg ttagtgtcaa gcctagctct
     5341 tttggggctg aaaagcaatc ctgcagtgct gaaaagcctc tcacaggcag ccgatgcggg
     5401 aagaggtgta ttagtcttga tagagaggct gcaaatagca ggaaacgtga gcagagactc
     5461 cctggtgtct gaaacacagg ccagatgggc cctcgagcag gaaacagcta tgaccatgat
     5521 tacgccaagc tatcaacttt gtatagaaaa gttggcacgc gcagattccg tgtgggcctg
     5581 gtaatatctt accattttaa gctattttaa gattagggaa aaaaattaaa taaaactact
     5641 ttaaaattac tttccttaaa aagcaacata tttatttagt ttttgtttaa ctggctcaag
     5701 attgtaatat tttactttta catgtttttt taagtaccta aatgtgttca aaagtaaagt
     5761 atgttattgt tttttttttt taatgttagc taagctttaa attgtaatat attactttta
     5821 aaagtgactt tttaaaaatg taaataaaaa agtaactata aatgtgtaat gcatgttatt
     5881 tctgtgggtt tttaattttc atctcaactg ggcttacaat tttaacataa taaaaatcaa
     5941 atcaaatcaa atcaggccct tatttgtcac atacactctt gtatactgaa ataaactaaa
     6001 tgttgagaaa aatttaaata caccaaaata acgcatacct tttttgtaat tacctaagta
     6061 aaacatttag ttcctttttt aagtgactca agattgtaat ctattaattg taaaagataa
     6121 aactagtgat caatctgacg tgcttctatg cagcctgtga gattaattgt gatgtttaat
     6181 tattaaacaa tttaaatgga tcaaacattt cacatgtgct tattgtgtcc aacaaaacca
     6241 gcagaagcac ttaaaaatat tgcatgacaa catgagtttt taatgtaaat tatatatact
     6301 tcctaaacga agttaatcat atttctaaaa taataatgtc attgaaatat ttaaataaat
     6361 gataagcttt agttgattca acataaaatt gaaagcaata aggacttttt ttttttacag
     6421 cggtgaagca aaagcatgta ataatcatat aatataataa ataaataaat aaataatggc
     6481 tcttggtgca aatatgaaca ctttaaataa atatagaata aatatgatca ataagtcata
     6541 acgatgtgtt ttatgttgat tataatttaa acatgtttct aacgcaagtt ttaaaaactg
     6601 ttttaaagta ttattttaag ttataagatc aatttgactc aacattttgt agtgaaaata
     6661 tgttgtgtaa aattaatata atatacacta taaaaatgca gggttccaca caatacatta
     6721 atgttgtccc aacacaaatc gattaagtta acaaatatat gtggattgaa cataaaaaat
     6781 taagttgtcc caatgaaatc tcaagaattg tgttgtttca gctcatttta aataagtagt
     6841 tttaacaagt aaaaaataac atatttttga gtgtgtatat atatatatta caaaatataa
     6901 taatataaaa tgtaaaagat ataaaccatg ctacatatat ttaagagcta ttttgcacat
     6961 tacttttttg caagccattt tagtgttttt tttacaaata tatattagct aaatatacaa
     7021 tatcttaaat atacaattaa tcagacatgc aattaatgca tttaacctac ataaaaatac
     7081 atgatcaaca tatgttttat gctgattaca actaaatcaa acgtaatgaa attaatttat
     7141 aaaagcgatt taaatatgtc aatattttaa gttaaaatca cttgaaacct actataaatg
     7201 aacttaaatt aaaaaaatct cctttaattc caatgcaaca gtgttgtttt ttcagctgag
     7261 gacccccggt gcagatggtt ggcccccggc ccgctattct aaggaagcct ttcttaattt
     7321 tagggcggcg ctgcatgcgc tcagatcaga ttcacgggtg gagagggtta ttttctcttt
     7381 tttatttttt cttgcctgct catatttggg cctgtgtgaa ccccaggctc cgcctcggcc
     7441 cccaggagcc gctcttattt aaaggggatc ctcgccttca gtcttcgtcc cagtggaagc
     7501 atcagtcttg tgataacaac ttggtgagtc ctacagacca gcgctcgact ctaggcttta
     7561 aatgcattga tttctctgga ttagctaccg ggagagtttt aactttgctt attagtcttg
     7621 tgctcgaact gctaggttta aagagtgttt tctctaatgc atgcatgaag cgacagctga
     7681 aaaccagaaa tagagcgttt gtcctcctgt taaactctag cctttctttg gcatttgtca
     7741 gtccgagtct tcagctgccg gcaggagatt gggtttcagt aacacttgct gagcacaaac
     7801 tacacgtgct ttcttcagat ttaattcatt attaatcatt ctagatttaa atgatgttct
     7861 tactcattgc tgattatttt agccttgcgg agcagattaa atgagcacgt gtttcattaa
     7921 gattgattta aatataatta atatttcacc actgtagttt taaaattgcc ttctaagtca
     7981 tggttgattt ttaataggta tcggctctaa ttagattact attttggttt ttaaatgtta
     8041 tttctatttc atttgtaggc tcgtttttaa tgtgatttta ttagattcta gttttaaaat
     8101 catctcttag ccttaatgag cagactgaat gaacatgttt cattaagatt aacttcatta
     8161 taatgaatta tttaatcatt ctagtgggtt ataaatcagc tctatttaaa ttaattgtcg
     8221 tgtttatttc tatttaattt ttgattattt tttcagggtc gtttttatgt gattttttcc
     8281 cgtttctaat tttaaactaa ttgtttgatt tttatatgat tcgactcttt ttaaatgaat
     8341 tgttttgctt tttttattga gcatatatat attatacaat ttcaatcaac aattgttgtg
     8401 ctttttaaat gttgtttcta tttcatttct agggtcgatt ttattcgatt ctagttttaa
     8461 aatcgtgatt tttaacttct aaccttactg agcagactga atgaacatgt ttcatcaaga
     8521 ttaacttcat tatagttaat tattgaatcg tgctagtggt ttataaatca gctctattta
     8581 aattaatcat catcattatt tctatttaat ttttattatt tctttaggat tatttttatg
     8641 tgattttttt cacgttctag ttttaaactc aatgctcatt actgctcacc cttgctgagc
     8701 agtctttcgt taagattaaa ttaattaata accattctag ttttaaaaca gtgtcttaag
     8761 gcatagctga tattttacaa gtttactatt taatatatct cctttctttt tttagggtcg
     8821 atttcatctg atttgattaa tttttattat cttcactaag ctttttgttc taggttttat
     8881 tctcagtatt attactgtta tatttgaact gaatttctcc tcatagttta ttcattttat
     8941 ttatttattt tttgtttatt cgctattttg tattttagtt tttaattaga aattttatta
     9001 gaatcgtttt gaactagttt ttatgcactc taacattaat attaaatcat aactattatt
     9061 tttattataa tttatttgtg tttatctttt tgtgtagctt tcatgtgttt ttattttagt
     9121 tttattatgt ttgcattgaa ttttttggac tagtttatat aacgatatac agaaaagcca
     9181 tgacaacaaa tgtttttata atacctatta agtataagct aatcagattt ccactattct
     9241 gttcaaagtt aacattatta gccattctga tggatcattt gacatttgtc atatgtcttt
     9301 atgaacactg ctctttcaga tgccactttt aaagctgttt tttacagtaa atgaacgttt
     9361 attattatga cattatttcc taaggcctct agagtacagc aggatgtggc gtgtgcatta
     9421 ccgtgtaatt atgctgactg taaatgtgca tttatagact gtcagcatgt ctgactgtgc
     9481 gctgttgtga ttggtcctgc acagaccaag
//
