LOCUS       sequence-274118-        5575 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of zebrafish GFP-tagged Rab33bb in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5575)
  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 5575)
  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-Rab33bb in pCSDEST2
FEATURES             Location/Qualifiers
     source          1..5575
                     /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"
     protein_bind    108..132
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      133..142
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(139..141,142..144,145..855)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(139..141,142..144,145..855)
                     /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(184..205)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(445..464)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     792..813
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(857..881)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1564..1584)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    1624..1758
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1648..1667)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1702..1721
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1871..1891)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1871..1889)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1910..1926)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1910..1926)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1923..1945)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1934..1950
                     /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(1958..1964,1965..1982,1983..1988))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2003..2024
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2141..2158)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2312..2900)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2392..2411)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3071..3862,3863..3931))
                     /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     3694..3713
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3932..4036)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4062..4517)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4199..4220)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4411..4430
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4591..5575
                     /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 tttttgcagg atcccatcga ttcgaattca aggatcaaca agtttgtaca
      121 aaaaagcagg ctgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
      181 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      241 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      301 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
      361 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      421 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
      481 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
      541 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
      601 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
      661 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
      721 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
      781 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
      841 gacgagctgt acaagcaccc agctttcttg tacaaagtgg tggattcgtc tttagaaagc
      901 tccagctcct ctaacggccc cccgacgcgc tgctgtcgga cctgtaagat catcgtgatc
      961 ggggatgccg gtgtgggcaa gacctgtctg acacatcgat tctgcaccgg acaatttcct
     1021 accagaacag aggcgaccat cggggtggat tttcgagaga aacttcttca gatagacggc
     1081 gaaaaaataa aagtacagct atgggacacg gccggtcagg agcgcttccg taaaagcatg
     1141 gtgcagcact actatcgaaa tgtgcatggt atcgtcttcg tctttgacgt cacaaaccaa
     1201 tccagtttct gcaatctgcc tttgtggatg gacgagtgcc gtcagcactc tcttggtctg
     1261 gaaattccac gtgccctagt ttgcaacaag gctgacctga tgtcttcaaa tgcttctgtg
     1321 ctaatcgagc aggctcgtca tttggctgaa gtccacggga tgtcttttta tctcacttca
     1381 gccaaaggtc ttaaaggcga ccaggtggac agcatcttca tggctttagc acagaggctg
     1441 aagagccaga gaagggagag cgtaaatgga cgtgtgctag agtgcagagc tgagagctta
     1501 aaaatccagg cacgggttat aacagcccaa gagactaaaa agaaatggac ctgcatctgc
     1561 taacaacttt attatacata gttgcactcg agcctctaga actatagtga gtcgtattac
     1621 gtagatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
     1681 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata
     1741 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg
     1801 gaggtgtggg aggtttttta attcgcggcc gcggcgccaa tgcattgggc ccggtaccca
     1861 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt
     1921 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     1981 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     2041 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     2101 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     2161 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     2221 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     2281 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2341 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2401 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2461 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2521 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2581 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2641 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2701 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat
     2761 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2821 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     2881 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     2941 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3001 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3061 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3121 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3181 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     3241 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3301 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3361 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3421 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3481 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3541 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3601 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3661 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3721 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3781 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3841 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     3901 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     3961 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4021 aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat
     4081 tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga
     4141 aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc
     4201 agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac
     4261 cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc
     4321 gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg
     4381 gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag
     4441 ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc
     4501 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
     4561 ggtgcgggcc tcttcgctat tacgccagtc gaccatagcc aattcaatat ggcgtatatg
     4621 gactcatgcc aattcaatat ggtggatctg gacctgtgcc aattcaatat ggcgtatatg
     4681 gactcgtgcc aattcaatat ggtggatctg gaccccagcc aattcaatat ggcggacttg
     4741 gcaccatgcc aattcaatat ggcggacttg gcactgtgcc aactggggag gggtctactt
     4801 ggcacggtgc caagtttgag gaggggtctt ggccctgtgc caagtccgcc atattgaatt
     4861 ggcatggtgc caataatggc ggccatattg gctatatgcc aggatcaata tataggcaat
     4921 atccaatatg gccctatgcc aatatggcta ttggccaggt tcaatactat gtattggccc
     4981 tatgccatat agtattccat atatgggttt tcctattgac gtagatagcc cctcccaatg
     5041 ggcggtccca tataccatat atggggcttc ctaataccgc ccatagccac tcccccattg
     5101 acgtcaatgg tctctatata tggtctttcc tattgacgtc atatgggcgg tcctattgac
     5161 gtatatggcg cctcccccat tgacgtcaat tacggtaaat ggcccgcctg gctcaatgcc
     5221 cattgacgtc aataggacca cccaccattg acgtcaatgg gatggctcat tgcccattca
     5281 tatccgttct cacgccccct attgacgtca atgacggtaa atggcccact tggcagtaca
     5341 tcaatatcta ttaatagtaa cttggcaagt acattactat tggaaggacg ccagggtaca
     5401 ttggcagtac tcccattgac gtcaatggcg gtaaatggcc cgcgatggct gccaagtaca
     5461 tccccattga cgtcaatggg gaggggcaat gacgcaaatg ggcgttccat tgacgtaaat
     5521 gggcggtagg cgtgcctaat gggaggtcta tataagcaat gctcgtttag ggaac
//
