LOCUS       sequence-382139-        5413 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mRuby Rab35 in mammalian cells and zebrafish embryos.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5413)
  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 5413)
  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- mRuby-Rab35
FEATURES             Location/Qualifiers
     source          1..5413
                     /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"
     regulatory      88..97
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             94..804
                     /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    1465..1599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1489..1508)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1543..1562
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1712..1732)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1712..1730)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1751..1767)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1751..1767)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1764..1786)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1775..1791
                     /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(1799..1805,1806..1823,1824..1829))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1844..1865
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1982..1999)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2153..2741)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2233..2252)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2912..3703,3704..3772))
                     /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     3535..3554
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3773..3877)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3903..4358)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4040..4061)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4252..4271
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4429..5413
                     /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 tttttgcagc cgctagagcc accatggtgt ctaagggcga agagctgatc
      121 aaggaaaata tgcgtatgaa ggtggtcatg gaaggttcgg tcaacggcca ccaattcaaa
      181 tgcacaggtg aaggagaagg caatccgtac atgggaactc aaaccatgag gatcaaagtc
      241 atcgagggag gacccctgcc atttgccttt gacattcttg ccacgtcgtt catgtatggc
      301 agccgtactt ttatcaagta cccgaaaggc attcctgatt tctttaaaca gtcctttcct
      361 gagggtttta cttgggaaag agttacgaga tacgaagatg gtggagtcgt caccgtcatg
      421 caggacacca gccttgagga tggctgtctc gtttaccacg tccaagtcag aggggtaaac
      481 tttccctcca atggtcccgt gatgcagaag aagaccaagg gttgggagcc taatacagag
      541 atgatgtatc cagcagatgg tggtctgagg ggatacactc atatggcact gaaagttgat
      601 ggtggtggcc atctgtcttg ctctttcgta acaacttaca ggtcaaaaaa gaccgtcggg
      661 aacatcaaga tgcccggtat ccatgccgtt gatcaccgcc tggaaaggtt agaggaaagt
      721 gacaatgaaa tgttcgtagt acaacgcgaa cacgcagttg ccaagttcgc cgggcttggt
      781 ggtgggatgg acgagctgta caagcgagct caagcttcga tggcccggga ctacgaccac
      841 ctcttcaagc tgctcatcat cggcgacagc ggtgtgggca agagcagttt actgttgcgt
      901 tttgcagaca acactttctc aggcagctac atcaccacga tcggagtgga tttcaagatc
      961 cggaccgtgg agatcaacgg ggagaaggtg aagctgcaga tctgggacac agcggggcag
     1021 gagcgcttcc gcaccatcac ctccacgtat tatcggggga cccacggggt cattgtggtt
     1081 tacgacgtca ccagtgccga gtcctttgtc aacgtcaagc ggtggcttca cgaaatcaac
     1141 cagaactgtg atgatgtgtg ccgaatatta gtgggtaata agaatgacga ccctgagcgg
     1201 aaggtggtgg agacggaaga tgcctacaaa ttcgccgggc agatgggcat ccagttgttc
     1261 gagaccagcg ccaaggagaa tgtcaacgtg gaagagatgt tcaactgcat cacggagctg
     1321 gtcctccgag caaagaaaga caacctggca aaacagcagc agcaacaaca gaacgatgtg
     1381 gtgaagctca cgaagaacag taaacgaaag aaacgctgct gctaagaatt ctgcagtcga
     1441 aactatagtg agtcgtatta cgtagatcca gacatgataa gatacattga tgagtttgga
     1501 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     1561 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     1621 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aattcgcggc cgcggcgcca
     1681 atgcattggg cccggtaccc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg
     1741 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     1801 acatacgagc cgggagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     1861 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     1921 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     1981 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     2041 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     2101 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     2161 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     2221 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     2281 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     2341 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     2401 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     2461 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     2521 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     2581 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     2641 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     2701 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     2761 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     2821 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     2881 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     2941 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     3001 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     3061 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     3121 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     3181 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     3241 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     3301 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     3361 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     3421 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     3481 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     3541 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     3601 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     3661 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     3721 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     3781 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     3841 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     3901 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
     3961 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
     4021 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
     4081 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
     4141 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     4201 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     4261 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     4321 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
     4381 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagt cgaccgccaa
     4441 ttcaatatgg cgtatatgga ctcatgccaa ttcaatatgg tggatctgga cctgtgccaa
     4501 ttcaatatgg cgtatatgga ctcgtgccaa ttcaatatgg tggatctgga ccccagccaa
     4561 ttcaatatgg cggacttggc accatgccaa ttcaatatgg cggacctggc actgtgccaa
     4621 ctggggaggg gtctacttgg cacggtgcca agtttgagga ggggtcttgg ccctgtgcca
     4681 agtccgccat attgaattgg catggtgcca ataatggcgg ccatattggc tatatgccag
     4741 gatcaatata taggcaatat ccaatatggc cctatgccaa tatggctatt ggccaggttc
     4801 aatactatgt attggcccta tgccatatag tattccatat atgggttttc ctattgacgt
     4861 agatagcccc tcccaatggg cggtcccata taccatatat ggggcttcct aataccgccc
     4921 atagccactc ccccattgac gtcaatggtc tctatatatg gtctttccta ttgacgtcat
     4981 atgggcggtc ctattgacgt atatggcgcc tcccccattg acgtcaatta cggtaaatgg
     5041 cccgcctggc tcaatgccca ttgacgtcaa taggaccacc caccattgac gtcaatggga
     5101 tggctcattg cccattcata tccgttctca cgccccctat tgacgtcaat gacggtaaat
     5161 ggcccacttg gcagtacatc aatatctatt aatagtaact tggcaagtac attactattg
     5221 gaagtacgcc agggtacatt ggcagtactc ccattgacgt caatggcggt aaatggcccg
     5281 cgatggctgc caagtacatc cccattgacg tcaatgggga ggggcaatga cgcaaatggg
     5341 cgttccattg acgtaaatgg gcggtaggcg tgcctaatgg gaggtctata taagcaatgc
     5401 tcgtttaggg aac
//
