LOCUS       sequence-382133-        4849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Lifeact with mRuby which labels F-actin in mammalian cells
            and zebrafish embryos.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4849)
  AUTHORS   Aljiboury AA, Ingram E, Krishnan N, Ononiwu F, Pal D, Manikas J, 
            Taveras C, Hall NA, Da Silva J, Freshour J, Hehnly H
  TITLE     Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during 
            zebrafish left-right organizer development.
  JOURNAL   PLoS Genet. 2023 May 15;19(5):e1010765. doi: 
            10.1371/journal.pgen.1010765. eCollection 2023 May.
  PUBMED    37186603
REFERENCE   2  (bases 1 to 4849)
  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: "PLoS 
            Genet."; date: "2023-05-15"; volume: "19(5):e1010765. doi"; pages: "
            10.1371/journal.pgen.1010765. eCollection 2023 May"
COMMENT     Sequence Label: pCS2-Lifeact-mRuby
FEATURES             Location/Qualifiers
     source          1..4849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             80..130
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     regulatory      146..155
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             152..862
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     polyA_signal    901..1035
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(925..944)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     979..998
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1148..1168)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1148..1166)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1187..1203)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1187..1203)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1200..1222)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1211..1227
                     /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(1235..1241,1242..1259,1260..1265))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1280..1301
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1418..1435)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1589..2177)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1669..1688)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2348..3139,3140..3208))
                     /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     2971..2990
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3209..3313)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3339..3794)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3476..3497)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3688..3707
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3865..4849
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcaga tgggcgtggc cgacttgatc aagaagttcg agtccatctc
      121 caaggaggag ggggatccac cggtcgccac catggtgtct aagggcgaag agctgatcaa
      181 ggaaaatatg cgtatgaagg tggtcatgga aggttcggtc aacggccacc aattcaaatg
      241 cacaggtgaa ggagaaggca atccgtacat gggaactcaa accatgagga tcaaagtcat
      301 cgagggagga cccctgccat ttgcctttga cattcttgcc acgtcgttca tgtatggcag
      361 ccgtactttt atcaagtacc cgaaaggcat tcctgatttc tttaaacagt cctttcctga
      421 gggttttact tgggaaagag ttacgagata cgaagatggt ggagtcgtca ccgtcatgca
      481 ggacaccagc cttgaggatg gctgtctcgt ttaccacgtc caagtcagag gggtaaactt
      541 tccctccaat ggtcccgtga tgcagaagaa gaccaagggt tgggagccta atacagagat
      601 gatgtatcca gcagatggtg gtctgagggg atacactcat atggcactga aagttgatgg
      661 tggtggccat ctgtcttgct ctttcgtaac aacttacagg tcaaaaaaga ccgtcgggaa
      721 catcaagatg cccggtatcc atgccgttga tcaccgcctg gaaaggttag aggaaagtga
      781 caatgaaatg ttcgtagtac aacgcgaaca cgcagttgcc aagttcgccg ggcttggtgg
      841 tgggatggac gagctgtaca agtaactgca gtctagaact atagtgagtc gtattacgta
      901 gatccagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
      961 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     1021 tgcaataaac aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag
     1081 gtgtgggagg ttttttaatt cgcggccgcg gcgccaatgc attgggcccg gtacccagct
     1141 tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc
     1201 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccggg agcataaagt
     1261 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     1321 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     1381 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     1441 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     1501 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     1561 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     1621 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     1681 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     1741 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     1801 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     1861 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     1921 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     1981 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     2041 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     2101 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     2161 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     2221 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     2281 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     2341 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     2401 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     2461 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     2521 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     2581 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     2641 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     2701 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     2761 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     2821 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     2881 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     2941 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     3001 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     3061 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     3121 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     3181 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     3241 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     3301 taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt
     3361 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     3421 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     3481 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     3541 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     3601 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     3661 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     3721 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     3781 gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
     3841 gcgggcctct tcgctattac gccagtcgac cgccaattca atatggcgta tatggactca
     3901 tgccaattca atatggtgga tctggacctg tgccaattca atatggcgta tatggactcg
     3961 tgccaattca atatggtgga tctggacccc agccaattca atatggcgga cttggcacca
     4021 tgccaattca atatggcgga cctggcactg tgccaactgg ggaggggtct acttggcacg
     4081 gtgccaagtt tgaggagggg tcttggccct gtgccaagtc cgccatattg aattggcatg
     4141 gtgccaataa tggcggccat attggctata tgccaggatc aatatatagg caatatccaa
     4201 tatggcccta tgccaatatg gctattggcc aggttcaata ctatgtattg gccctatgcc
     4261 atatagtatt ccatatatgg gttttcctat tgacgtagat agcccctccc aatgggcggt
     4321 cccatatacc atatatgggg cttcctaata ccgcccatag ccactccccc attgacgtca
     4381 atggtctcta tatatggtct ttcctattga cgtcatatgg gcggtcctat tgacgtatat
     4441 ggcgcctccc ccattgacgt caattacggt aaatggcccg cctggctcaa tgcccattga
     4501 cgtcaatagg accacccacc attgacgtca atgggatggc tcattgccca ttcatatccg
     4561 ttctcacgcc ccctattgac gtcaatgacg gtaaatggcc cacttggcag tacatcaata
     4621 tctattaata gtaacttggc aagtacatta ctattggaag tacgccaggg tacattggca
     4681 gtactcccat tgacgtcaat ggcggtaaat ggcccgcgat ggctgccaag tacatcccca
     4741 ttgacgtcaa tggggagggg caatgacgca aatgggcgtt ccattgacgt aaatgggcgg
     4801 taggcgtgcc taatgggagg tctatataag caatgctcgt ttagggaac
//
