LOCUS       sequence-220532-        9507 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Muscle specific zebrafish expression of mKate2 fused to dominant 
            negative Rac1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9507)
  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 9507)
  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-Rac1-DN
FEATURES             Location/Qualifiers
     source          1..9507
                     /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(1331..1351)
                     /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(1359..1375)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    1391..1525
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1415..1434)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1469..1488
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(1964..2151,2152..2163)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(2197..2213)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2197..2213)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2250..2270)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2250..2268)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2289..2305)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2302..2324)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2313..2329
                     /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(2337..2343,2344..2361,2362..2367))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2382..2403
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2520..2537)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2691..3279)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2771..2790)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3450..4241,4242..4310))
                     /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     4073..4092
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4311..4415)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4578..4599)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     misc_feature    join(4948..4959,4960..5122)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     5495..5511
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5531..5551
                     /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 ggctcaggcc ataaagtgtg tggtcgtcgg
      781 ggacggggct gtgggaaaaa actgccttct gatcagctac accaccaatg cttttcctgg
      841 ggaatacatt cccactgtgt ttgataatta ttctgcaaat gtgatggtcg atgggaaacc
      901 agtaaacctg ggattgtggg atacagcagg acaagaggat tatgacaggc ttcgaccgct
      961 ttcctaccct cagacggatg tgttcctgat ctgcttctct cttgtgagtc cggcctcatt
     1021 tgaaaacgtc cgtgcaaagt ggtatccgga ggtcagacat cattgcccta acactccaat
     1081 aattcttgtt gggactaagc ttgatttgcg agatgacaag gacaccatcg aaaagttaaa
     1141 ggagaagaaa ctcactccca tcacctaccc tcaaggcctt gctatggcca aagaaatagg
     1201 agctgtgaag tacctggagt gctctgcctt aacccaacgc ggccttaaaa ctgtgttcga
     1261 tgaagccatc cgtgcagtgc tgtgtccacc tccggtgaag aggaggagga gaagatgtct
     1321 ccttctgtga caactttatt atacatagtt gataattcac tggccgtcgt tttacggtac
     1381 catcgatgat gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag
     1441 aatgcagtga aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac
     1501 cattataagc tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt
     1561 tcagggggag gtgtgggagg ttttttaaag caagtaaaac ctctacaaat gtggtatggc
     1621 tgattatgat cctctagatc agatctgcga agatacggcc acgggtgctc ttgatcctgt
     1681 ggctgatttt ggactgtgct gctcgcagct gctgatgaat cacatacttc ctccattttc
     1741 ttccactgat tgactgttat aatttcccta atttccaggt caaggtgctg tgcattgtgg
     1801 taatagatgt gacatgacgt cacttccaaa ggaccaatga acatgtctga ccaatttcat
     1861 ataatgtgaa aacgattttc ataggcagaa taaataacat ttaaattaaa ctgggcatca
     1921 gcgcaattca attggtttgg taatagcaag ggaaaataga atgaagtgat ctccaaaaaa
     1981 taagtacttt ttgactgtaa ataaaattgt aaggagtaaa aagtactttt ttttctaaaa
     2041 aaatgtaatt aagtaaaagt aaaagtattg atttttaatt gtactcaagt aaagtaaaaa
     2101 tccccaaaaa taatacttaa gtacagtaat caagtaaaat tactcaagta ctttacacct
     2161 ctggttcttg accccctacc ttcagcaagc ccagcagatc cactagttct agagcggccg
     2221 ccaccgcggt ggagctccag cttttgttcc ctttagtgag ggttaattgc gcgcttggcg
     2281 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     2341 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     2401 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     2461 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     2521 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     2581 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     2641 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     2701 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     2761 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     2821 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     2881 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     2941 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     3001 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     3061 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     3121 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     3181 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     3241 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     3301 acggggtctg acgctcagtg gaacgaaaac tcacgttaag gcattttggt catgagatta
     3361 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     3421 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3481 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3541 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3601 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3661 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3721 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3781 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3841 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3901 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3961 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     4021 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     4081 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     4141 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4201 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4261 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4321 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4381 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4441 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
     4501 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
     4561 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
     4621 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc acgctggcta agaactcatc
     4681 agcctccccg gtccatctac ccacgtacca atgcaccaat tggccacaat gacggctact
     4741 acatggtgcc attccttcct ctttatagga atggagacta cctcctgtcc aacaatgctc
     4801 ttggatacga gtacgcctac ctgttggacc caggtcattg cacaacacca gaaatgccct
     4861 ctgatctgca aaagacgtga atatctgttc agacacccat atccactctg ttccacacag
     4921 gtcagaggtt tgtccaggag ttcttgacag aggtgtaaaa agtactcaaa aattttactc
     4981 aagtgaaagt acaagtactt agggaaaatt ttactcaatt aaaagtaaaa gtatctggct
     5041 agaatcttac ttgagtaaaa gtaaaaaagt actccattaa aattgtactt gagtattaag
     5101 gaagtaaaag taaaagcaag aaagaaaact agagattctt gtttaagctt ttaatctcaa
     5161 aaaacattaa atgaaatgca tacaaggttt tatcctgctt tagaactgtt tgtatttaat
     5221 tatcaaacta taagacagac aatctaatgc cagtacacgc tactcaaagt tgtaaaacct
     5281 cagatttaac ttcagtagaa gctgattctc aaaattgtta gtgtcaagcc tagctctttt
     5341 ggggctgaaa agcaatcctg cagtgctgaa aagcctctca caggcagccg atgcgggaag
     5401 aggtgtatta gtcttgatag agaggctgca aatagcagga aacgtgagca gagactccct
     5461 ggtgtctgaa acacaggcca gatgggccct cgagcaggaa acagctatga ccatgattac
     5521 gccaagctat caactttgta tagaaaagtt ggcacgcgca gattccgtgt gggcctggta
     5581 atatcttacc attttaagct attttaagat tagggaaaaa aattaaataa aactacttta
     5641 aaattacttt ccttaaaaag caacatattt atttagtttt tgtttaactg gctcaagatt
     5701 gtaatatttt acttttacat gtttttttaa gtacctaaat gtgttcaaaa gtaaagtatg
     5761 ttattgtttt ttttttttaa tgttagctaa gctttaaatt gtaatatatt acttttaaaa
     5821 gtgacttttt aaaaatgtaa ataaaaaagt aactataaat gtgtaatgca tgttatttct
     5881 gtgggttttt aattttcatc tcaactgggc ttacaatttt aacataataa aaatcaaatc
     5941 aaatcaaatc aggcccttat ttgtcacata cactcttgta tactgaaata aactaaatgt
     6001 tgagaaaaat ttaaatacac caaaataacg catacctttt ttgtaattac ctaagtaaaa
     6061 catttagttc cttttttaag tgactcaaga ttgtaatcta ttaattgtaa aagataaaac
     6121 tagtgatcaa tctgacgtgc ttctatgcag cctgtgagat taattgtgat gtttaattat
     6181 taaacaattt aaatggatca aacatttcac atgtgcttat tgtgtccaac aaaaccagca
     6241 gaagcactta aaaatattgc atgacaacat gagtttttaa tgtaaattat atatacttcc
     6301 taaacgaagt taatcatatt tctaaaataa taatgtcatt gaaatattta aataaatgat
     6361 aagctttagt tgattcaaca taaaattgaa agcaataagg actttttttt tttacagcgg
     6421 tgaagcaaaa gcatgtaata atcatataat ataataaata aataaataaa taatggctct
     6481 tggtgcaaat atgaacactt taaataaata tagaataaat atgatcaata agtcataacg
     6541 atgtgtttta tgttgattat aatttaaaca tgtttctaac gcaagtttta aaaactgttt
     6601 taaagtatta ttttaagtta taagatcaat ttgactcaac attttgtagt gaaaatatgt
     6661 tgtgtaaaat taatataata tacactataa aaatgcaggg ttccacacaa tacattaatg
     6721 ttgtcccaac acaaatcgat taagttaaca aatatatgtg gattgaacat aaaaaattaa
     6781 gttgtcccaa tgaaatctca agaattgtgt tgtttcagct cattttaaat aagtagtttt
     6841 aacaagtaaa aaataacata tttttgagtg tgtatatata tatattacaa aatataataa
     6901 tataaaatgt aaaagatata aaccatgcta catatattta agagctattt tgcacattac
     6961 ttttttgcaa gccattttag tgtttttttt acaaatatat attagctaaa tatacaatat
     7021 cttaaatata caattaatca gacatgcaat taatgcattt aacctacata aaaatacatg
     7081 atcaacatat gttttatgct gattacaact aaatcaaacg taatgaaatt aatttataaa
     7141 agcgatttaa atatgtcaat attttaagtt aaaatcactt gaaacctact ataaatgaac
     7201 ttaaattaaa aaaatctcct ttaattccaa tgcaacagtg ttgttttttc agctgaggac
     7261 ccccggtgca gatggttggc ccccggcccg ctattctaag gaagcctttc ttaattttag
     7321 ggcggcgctg catgcgctca gatcagattc acgggtggag agggttattt tctctttttt
     7381 attttttctt gcctgctcat atttgggcct gtgtgaaccc caggctccgc ctcggccccc
     7441 aggagccgct cttatttaaa ggggatcctc gccttcagtc ttcgtcccag tggaagcatc
     7501 agtcttgtga taacaacttg gtgagtccta cagaccagcg ctcgactcta ggctttaaat
     7561 gcattgattt ctctggatta gctaccggga gagttttaac tttgcttatt agtcttgtgc
     7621 tcgaactgct aggtttaaag agtgttttct ctaatgcatg catgaagcga cagctgaaaa
     7681 ccagaaatag agcgtttgtc ctcctgttaa actctagcct ttctttggca tttgtcagtc
     7741 cgagtcttca gctgccggca ggagattggg tttcagtaac acttgctgag cacaaactac
     7801 acgtgctttc ttcagattta attcattatt aatcattcta gatttaaatg atgttcttac
     7861 tcattgctga ttattttagc cttgcggagc agattaaatg agcacgtgtt tcattaagat
     7921 tgatttaaat ataattaata tttcaccact gtagttttaa aattgccttc taagtcatgg
     7981 ttgattttta ataggtatcg gctctaatta gattactatt ttggttttta aatgttattt
     8041 ctatttcatt tgtaggctcg tttttaatgt gattttatta gattctagtt ttaaaatcat
     8101 ctcttagcct taatgagcag actgaatgaa catgtttcat taagattaac ttcattataa
     8161 tgaattattt aatcattcta gtgggttata aatcagctct atttaaatta attgtcgtgt
     8221 ttatttctat ttaatttttg attatttttt cagggtcgtt tttatgtgat tttttcccgt
     8281 ttctaatttt aaactaattg tttgattttt atatgattcg actcttttta aatgaattgt
     8341 tttgcttttt ttattgagca tatatatatt atacaatttc aatcaacaat tgttgtgctt
     8401 tttaaatgtt gtttctattt catttctagg gtcgatttta ttcgattcta gttttaaaat
     8461 cgtgattttt aacttctaac cttactgagc agactgaatg aacatgtttc atcaagatta
     8521 acttcattat agttaattat tgaatcgtgc tagtggttta taaatcagct ctatttaaat
     8581 taatcatcat cattatttct atttaatttt tattatttct ttaggattat ttttatgtga
     8641 tttttttcac gttctagttt taaactcaat gctcattact gctcaccctt gctgagcagt
     8701 ctttcgttaa gattaaatta attaataacc attctagttt taaaacagtg tcttaaggca
     8761 tagctgatat tttacaagtt tactatttaa tatatctcct ttcttttttt agggtcgatt
     8821 tcatctgatt tgattaattt ttattatctt cactaagctt tttgttctag gttttattct
     8881 cagtattatt actgttatat ttgaactgaa tttctcctca tagtttattc attttattta
     8941 tttatttttt gtttattcgc tattttgtat tttagttttt aattagaaat tttattagaa
     9001 tcgttttgaa ctagttttta tgcactctaa cattaatatt aaatcataac tattattttt
     9061 attataattt atttgtgttt atctttttgt gtagctttca tgtgttttta ttttagtttt
     9121 attatgtttg cattgaattt tttggactag tttatataac gatatacaga aaagccatga
     9181 caacaaatgt ttttataata cctattaagt ataagctaat cagatttcca ctattctgtt
     9241 caaagttaac attattagcc attctgatgg atcatttgac atttgtcata tgtctttatg
     9301 aacactgctc tttcagatgc cacttttaaa gctgtttttt acagtaaatg aacgtttatt
     9361 attatgacat tatttcctaa ggcctctaga gtacagcagg atgtggcgtg tgcattaccg
     9421 tgtaattatg ctgactgtaa atgtgcattt atagactgtc agcatgtctg actgtgcgct
     9481 gttgtgattg gtcctgcaca gaccaag
//
