LOCUS       sequence-325112-        5524 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of zebrafish GFP-tagged Rab8b in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5524)
  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 5524)
  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: EGFP-Rab8b in pCSDEST2
FEATURES             Location/Qualifiers
     source          1..5524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        64..1048
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1083..1101
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1083..1100
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    1156..1180
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1181..1190
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1187..1189,1190..1192,1193..1903)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1187..1189,1190..1192,1193..1903)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1232..1253)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1493..1512)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1840..1861
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1905..1929)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2564..2584)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2624..2758
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2648..2667)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2702..2721
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2871..2891)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2871..2889)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2910..2926)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2910..2926)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2923..2945)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2934..2950
                     /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(2958..2964,2965..2982,2983..2988))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3003..3024
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3141..3158)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3312..3900)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3392..3411)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4071..4862,4863..4931))
                     /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     4694..4713
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4932..5036)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5062..5517)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5199..5220)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5411..5430
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
       61 ccagtcgacc gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat
      121 ctggacctgt gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat
      181 ctggacccca gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac
      241 ctggcactgt gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt
      301 cttggccctg tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata
      361 ttggctatat gccaggatca atatataggc aatatccaat atggccctat gccaatatgg
      421 ctattggcca ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg
      481 ttttcctatt gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc
      541 ttcctaatac cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt
      601 tcctattgac gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc
      661 aattacggta aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca
      721 ttgacgtcaa tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg
      781 tcaatgacgg taaatggccc acttggcagt acatcaatat ctattaatag taacttggca
      841 agtacattac tattggaagt acgccagggt acattggcag tactcccatt gacgtcaatg
      901 gcggtaaatg gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc
      961 aatgacgcaa atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt
     1021 ctatataagc aatgctcgtt tagggaaccg ccattctgcc tggggacgtc ggagcaagct
     1081 tgatttaggt gacactatag aatacaagct acttgttctt tttgcaggat cccatcgatt
     1141 cgaattcaag gatcaacaag tttgtacaaa aaagcaggct gccaccatgg tgagcaaggg
     1201 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     1261 ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct
     1321 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct
     1381 gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1441 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg
     1501 caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1561 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1621 ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa
     1681 cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1741 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca
     1801 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1861 gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagcacccag ctttcttgta
     1921 caaagtggtg gcgaaaacgt acgactatct gttcaaactg ctgctcatcg gagacagcgg
     1981 tgtgggcaag acctgtctgc tgtttcgatt cagcgaggac gccttcaaca ccactttcat
     2041 ctccaccata ggaattgact tcaaaatcag aacaattgag ctggatggga aaaagatcaa
     2101 gcttcaaatc tgggatacgg cgggacagga gaggttcaga accatcacca ctgcatatta
     2161 cagaggagca atgggaatta tgctggtgta tgatatcaca aatgagaaat catttgacaa
     2221 catcaagaac tggatcagga acattgagga gcatgcatcg tcagatgtgg agcgaatgat
     2281 attgggtaac aggtgtgaca tgaacgacaa gagacaagtt tcaaaagaaa gaggagagaa
     2341 gctagctatt gattacggaa tcaagttctt ggaaaccagt gcaaaatcta gcacaaatgt
     2401 tgaagaggca tttgtcactc ttgctagaga catcatgacg agattaaaca ggaaaatgaa
     2461 tgagaataac ccttcagggg gaggaggagg aggagcagta aaaatcacag agagtcgatc
     2521 caagaagcca agcttcttcc gctgcattct gctctaaccc agccaacttt attatacata
     2581 gttgcactcg agcctctaga actatagtga gtcgtattac gtagatccag acatgataag
     2641 atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat gctttatttg
     2701 tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata aacaagttaa
     2761 caacaacaat tgcattcatt ttatgtttca ggttcagggg gaggtgtggg aggtttttta
     2821 attcgcggcc gcggcgccaa tgcattgggc ccggtaccca gcttttgttc cctttagtga
     2881 gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     2941 ccgctcacaa ttccacacaa catacgagcc gggagcataa agtgtaaagc ctggggtgcc
     3001 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3061 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3121 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3181 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3241 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3301 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3361 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3421 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3481 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3541 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3601 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     3661 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     3721 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     3781 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     3841 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     3901 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     3961 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4021 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4081 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4141 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4201 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4261 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4321 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4381 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4441 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4501 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4561 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4621 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     4681 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     4741 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     4801 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     4861 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     4921 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     4981 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5041 tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa
     5101 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
     5161 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
     5221 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
     5281 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
     5341 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
     5401 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
     5461 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc
     5521 attc
//
