LOCUS       sequence-325113-        5572 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 5572)
  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 5572)
  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..5572
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        36..1013
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
     promoter        1048..1066
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1048..1065
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    1121..1145
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1146..1155
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1152..1154,1155..1157,1158..1868)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1152..1154,1155..1157,1158..1868)
                     /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(1197..1218)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1458..1477)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1805..1826
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1870..1894)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2577..2597)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    2637..2771
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2661..2680)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2715..2734
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2884..2904)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2884..2902)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2923..2939)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2923..2939)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2936..2958)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2947..2963
                     /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(2971..2977,2978..2995,2996..3001))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3016..3037
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3154..3171)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3325..3913)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3405..3424)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4084..4875,4876..4944))
                     /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     4707..4726
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4945..5049)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5075..5530)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5212..5233)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5424..5443
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cggtgcgggc ctcttcgcta ttacgccagt cgaccgccaa ttcaatatgg cgtatatgga
       61 ctcatgccaa ttcaatatgg tggatctgga cctgtgccaa ttcaatatgg cgtatatgga
      121 ctcgtgccaa ttcaatatgg tggatctgga ccccagccaa ttcaatatgg cggacttggc
      181 accatgccaa ttcaatatgg cggacctggc actgtgccaa ctggggaggg gtctacttgg
      241 cacggtgcca agtttgagga ggggtcttgg ccctgtgcca agtccgccat attgaattgg
      301 catggtgcca ataatggcgg ccatattggc tatatgccag gatcaatata taggcaatat
      361 ccaatatggc cctatgccaa tatggctatt ggccaggttc aatactatgt attggcccta
      421 tgccatatag tattccatat atgggttttc ctattgacgt agatagcccc tcccaatggg
      481 cggtcccata taccatatat ggggcttcct aataccgccc atagccactc ccccattgac
      541 gtcaatggtc tctatatatg gtctttccta ttgacgtcat atgggcggtc ctattgacgt
      601 atatggcgcc tcccccattg acgtcaatta cggtaaatgg cccgcctggc tcaatgccca
      661 ttgacgtcaa taggaccacc caccattgac gtcaatggga tggctcattg cccattcata
      721 tccgttctca cgccccctat tgacgtcaat gacggtaaat ggcccacttg gcagtacatc
      781 aatatctatt aatagtaact tggcaagtac attactattg gaagtacgcc agggtacatt
      841 ggcagtactc ccattgacgt caatggcggt aaatggcccg cgatggctgc caagtacatc
      901 cccattgacg tcaatgggga ggggcaatga cgcaaatggg cgttccattg acgtaaatgg
      961 gcggtaggcg tgcctaatgg gaggtctata taagcaatgc tcgtttaggg aaccgccatt
     1021 ctgcctgggg acgtcggagc aagcttgatt taggtgacac tatagaatac aagctacttg
     1081 ttctttttgc aggatcccat cgattcgaat tcaaggatca acaagtttgt acaaaaaagc
     1141 aggctgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1201 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1261 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1321 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     1381 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1441 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1501 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1561 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     1621 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     1681 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1741 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     1801 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     1861 tgtacaagca cccagctttc ttgtacaaag tggtggattc gtctttagaa agctccagct
     1921 cctctaacgg ccccccgacg cgctgctgtc ggacctgtaa gatcatcgtg atcggggatg
     1981 ccggtgtggg caagacctgt ctgacacatc gattctgcac cggacaattt cctaccagaa
     2041 cagaggcgac catcggggtg gattttcgag agaaacttct tcagatagac ggcgaaaaaa
     2101 taaaagtaca gctatgggac acggccggtc aggagcgctt ccgtaaaagc atggtgcagc
     2161 actactatcg aaatgtgcat ggtatcgtct tcgtctttga cgtcacaaac caatccagtt
     2221 tctgcaatct gcctttgtgg atggacgagt gccgtcagca ctctcttggt ctggaaattc
     2281 cacgtgccct agtttgcaac aaggctgacc tgatgtcttc aaatgcttct gtgctaatcg
     2341 agcaggctcg tcatttggct gaagtccacg ggatgtcttt ttatctcact tcagccaaag
     2401 gtcttaaagg cgaccaggtg gacagcatct tcatggcttt agcacagagg ctgaagagcc
     2461 agagaaggga gagcgtaaat ggacgtgtgc tagagtgcag agctgagagc ttaaaaatcc
     2521 aggcacgggt tataacagcc caagagacta aaaagaaatg gacctgcatc tgctaacaac
     2581 tttattatac atagttgcac tcgagcctct agaactatag tgagtcgtat tacgtagatc
     2641 cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
     2701 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     2761 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
     2821 gggaggtttt ttaattcgcg gccgcggcgc caatgcattg ggcccggtac ccagcttttg
     2881 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
     2941 gtgaaattgt tatccgctca caattccaca caacatacga gccgggagca taaagtgtaa
     3001 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3061 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3121 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3181 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3241 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3301 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3361 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3421 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3481 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3541 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3601 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     3661 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     3721 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     3781 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     3841 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     3901 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     3961 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4021 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4081 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4141 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4201 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4261 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4321 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4381 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4441 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4501 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4561 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4621 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     4681 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     4741 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     4801 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     4861 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     4921 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     4981 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5041 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     5101 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     5161 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     5221 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     5281 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     5341 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     5401 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     5461 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     5521 cagggcgcgt cccattcgcc attcaggctg cgcaactgtt gggaagggcg at
//
