LOCUS       sequence-325139-        5524 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of zebrafish GFP-tagged Rab32b 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-Rab32b
FEATURES             Location/Qualifiers
     source          1..5524
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        889..907
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     889..906
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    962..986
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      987..996
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(993..995,996..998,999..1709)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(993..995,996..998,999..1709)
                     /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(1038..1059)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1299..1318)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1646..1667
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1711..1735)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2370..2390)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2430..2564
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2454..2473)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2508..2527
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2677..2697)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2677..2695)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2716..2732)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2716..2732)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2729..2751)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2740..2756
                     /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(2764..2770,2771..2788,2789..2794))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2809..2830
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2947..2964)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3118..3706)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3198..3217)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3877..4668,4669..4737))
                     /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     4500..4519
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4738..4842)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4868..5323)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5005..5026)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5217..5236
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        join(5394..5524,1..854)
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 attcaatatg gcggacttgg caccatgcca attcaatatg gcggacctgg cactgtgcca
       61 actggggagg ggtctacttg gcacggtgcc aagtttgagg aggggtcttg gccctgtgcc
      121 aagtccgcca tattgaattg gcatggtgcc aataatggcg gccatattgg ctatatgcca
      181 ggatcaatat ataggcaata tccaatatgg ccctatgcca atatggctat tggccaggtt
      241 caatactatg tattggccct atgccatata gtattccata tatgggtttt cctattgacg
      301 tagatagccc ctcccaatgg gcggtcccat ataccatata tggggcttcc taataccgcc
      361 catagccact cccccattga cgtcaatggt ctctatatat ggtctttcct attgacgtca
      421 tatgggcggt cctattgacg tatatggcgc ctcccccatt gacgtcaatt acggtaaatg
      481 gcccgcctgg ctcaatgccc attgacgtca ataggaccac ccaccattga cgtcaatggg
      541 atggctcatt gcccattcat atccgttctc acgcccccta ttgacgtcaa tgacggtaaa
      601 tggcccactt ggcagtacat caatatctat taatagtaac ttggcaagta cattactatt
      661 ggaagtacgc cagggtacat tggcagtact cccattgacg tcaatggcgg taaatggccc
      721 gcgatggctg ccaagtacat ccccattgac gtcaatgggg aggggcaatg acgcaaatgg
      781 gcgttccatt gacgtaaatg ggcggtaggc gtgcctaatg ggaggtctat ataagcaatg
      841 ctcgtttagg gaaccgccat tctgcctggg gacgtcggag caagcttgat ttaggtgaca
      901 ctatagaata caagctactt gttctttttg caggatccca tcgattcgaa ttcaaggatc
      961 aacaagtttg tacaaaaaag caggctgcca ccatggtgag caagggcgag gagctgttca
     1021 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1081 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1141 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     1201 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1261 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1321 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1381 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     1441 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     1501 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     1561 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     1621 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     1681 tcactctcgg catggacgag ctgtacaagc acccagcttt cttgtacaaa gtggtgagga
     1741 aaatgtcagg agactcagag ttaggatgca ctgaacattt atttaaagtt ttggtgattg
     1801 gagaccataa agttgggaag agcagctttg ttaaactcta tgtgaggaat cagttttacg
     1861 aggagttgaa aaccagtata ggtgtggatt tttctatgaa ggttatcgat tgggacgctc
     1921 acacacgagt gagattgcag ttttgggata tagctggtca agagcgtgtg cgaggactga
     1981 atcgtgtgta cttcagggga cccagtggtg cttttgtagt atacgacctg acaagcggct
     2041 catcgctgga tggggctctg aactggaaac gtgaactcga ttgtcaagta atgttgaaaa
     2101 atggccatcc gatccctgct gttctgctgg ctaataaatg tgatgagact aaagtgagcc
     2161 ggaggaacgc atcccttctg gagaagctct gccaggagaa gggttttact gggtgctttc
     2221 agacttcagc aaaggagaat atcaatgtgg atgaagctgc taaatttctt gtgaagcaca
     2281 ttctccaaat taacgaagaa tcaatgaaag aagaagagca gaagattcac ctcaaaccac
     2341 acacaaacac aaactcggtc tgctgctgac aactttatta tacatagttg cactcgagcc
     2401 tctagaacta tagtgagtcg tattacgtag atccagacat gataagatac attgatgagt
     2461 ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg
     2521 ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac aacaattgca
     2581 ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaattc gcggccgcgg
     2641 cgccaatgca ttgggcccgg tacccagctt ttgttccctt tagtgagggt taattgcgcg
     2701 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2761 acacaacata cgagccggga gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     2821 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     2881 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     2941 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3001 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3061 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3121 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3181 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3241 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3301 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3361 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3421 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3481 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3541 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3601 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3661 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3721 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3781 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3841 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3901 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3961 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     4021 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     4081 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     4141 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4201 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4261 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4321 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4381 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4441 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4501 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4561 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4621 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4681 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4741 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4801 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4861 gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     4921 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     4981 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     5041 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     5101 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     5161 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5221 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5281 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gccattcagg
     5341 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagtcgacc
     5401 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
     5461 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
     5521 gcca
//
