LOCUS       sequence-325082-        5512 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of zebrafish GFP-tagged Rab8a in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5512)
  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 5512)
  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-Rab8a in pCSDEST2
FEATURES             Location/Qualifiers
     source          1..5512
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        51..1035
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1070..1088
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1070..1087
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    1143..1167
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1168..1177
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1174..1176,1177..1179,1180..1890)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1174..1176,1177..1179,1180..1890)
                     /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(1219..1240)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1480..1499)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1827..1848
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1892..1916)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2539..2559)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2599..2733
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2623..2642)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2677..2696
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2846..2866)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2846..2864)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2885..2901)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2885..2901)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2898..2920)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2909..2925
                     /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(2933..2939,2940..2957,2958..2963))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2978..2999
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3116..3133)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3287..3875)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3367..3386)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4046..4837,4838..4906))
                     /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     4669..4688
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4907..5011)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5037..5492)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5174..5195)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5386..5405
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgaccgcc
       61 aattcaatat ggcgtatatg gactcatgcc aattcaatat ggtggatctg gacctgtgcc
      121 aattcaatat ggcgtatatg gactcgtgcc aattcaatat ggtggatctg gaccccagcc
      181 aattcaatat ggcggacttg gcaccatgcc aattcaatat ggcggacctg gcactgtgcc
      241 aactggggag gggtctactt ggcacggtgc caagtttgag gaggggtctt ggccctgtgc
      301 caagtccgcc atattgaatt ggcatggtgc caataatggc ggccatattg gctatatgcc
      361 aggatcaata tataggcaat atccaatatg gccctatgcc aatatggcta ttggccaggt
      421 tcaatactat gtattggccc tatgccatat agtattccat atatgggttt tcctattgac
      481 gtagatagcc cctcccaatg ggcggtccca tataccatat atggggcttc ctaataccgc
      541 ccatagccac tcccccattg acgtcaatgg tctctatata tggtctttcc tattgacgtc
      601 atatgggcgg tcctattgac gtatatggcg cctcccccat tgacgtcaat tacggtaaat
      661 ggcccgcctg gctcaatgcc cattgacgtc aataggacca cccaccattg acgtcaatgg
      721 gatggctcat tgcccattca tatccgttct cacgccccct attgacgtca atgacggtaa
      781 atggcccact tggcagtaca tcaatatcta ttaatagtaa cttggcaagt acattactat
      841 tggaagtacg ccagggtaca ttggcagtac tcccattgac gtcaatggcg gtaaatggcc
      901 cgcgatggct gccaagtaca tccccattga cgtcaatggg gaggggcaat gacgcaaatg
      961 ggcgttccat tgacgtaaat gggcggtagg cgtgcctaat gggaggtcta tataagcaat
     1021 gctcgtttag ggaaccgcca ttctgcctgg ggacgtcgga gcaagcttga tttaggtgac
     1081 actatagaat acaagctact tgttcttttt gcaggatccc atcgattcga attcaaggat
     1141 caacaagttt gtacaaaaaa gcaggctgcc accatggtga gcaagggcga ggagctgttc
     1201 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
     1261 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gttcatctgc
     1321 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac ctacggcgtg
     1381 cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
     1441 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
     1501 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
     1561 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
     1621 aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt caagatccgc
     1681 cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc
     1741 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc cgccctgagc
     1801 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     1861 atcactctcg gcatggacga gctgtacaag cacccagctt tcttgtacaa agtggtggcg
     1921 aagacctacg attatttgtt taaactgctg ttaatcgggg attccggagt cgggaagacc
     1981 tgtgtgctgt tcagattctc agaggatgcc tttaactcga catttatctc gacgataggt
     2041 atcgatttca agatcaggac aatagaacta gatggcaaga agataaagtt acagatatgg
     2101 gacacggcag gacaggaacg atttcggacg atcacgacag catattacag aggtgcaatg
     2161 ggtattatgc tggtttatga tatcaccaat gagaagtcat ttgacaacat caaaaactgg
     2221 atccgaaaca tagaggagca tgcatcagct gatgtggaaa agatgatttt ggggaacaaa
     2281 tgtgacatta atgagaagag gcaggtgtct aaagacagag gcgagaagct ggcgttagag
     2341 tacggcatca agttcatgga gacaagtgca aaggctaata tcaacgttga gaatgctttc
     2401 ttaacactcg ccagagacat aaaagcaaag atggacacaa aattggaggg aaacaatcct
     2461 cagggcagca accacggtgt gaaaattacc actgaacagc cgaaaaagag cagcttcttt
     2521 cgctgtgtgc tactgtgaca actttattat acatagttgc actcgagcct ctagaactat
     2581 agtgagtcgt attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc
     2641 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     2701 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     2761 tttcaggttc agggggaggt gtgggaggtt ttttaattcg cggccgcggc gccaatgcat
     2821 tgggcccggt acccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat
     2881 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     2941 gagccgggag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     3001 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     3061 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3121 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3181 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3241 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3301 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     3361 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     3421 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     3481 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     3541 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     3601 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     3661 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     3721 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     3781 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     3841 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     3901 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     3961 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4021 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4081 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4141 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4201 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4261 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4321 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     4381 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     4441 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     4501 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     4561 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     4621 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     4681 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     4741 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     4801 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     4861 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     4921 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     4981 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     5041 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     5101 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     5161 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     5221 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     5281 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     5341 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     5401 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     5461 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tg
//
