LOCUS       sequence-333285-        5391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Rab6B fused to APEX2 and a Flag tag in N-ter under control of a weak
            L30 promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5391)
  AUTHORS   Raphael Gaudin Lab
  TITLE     Generation of APEX2-fused Rab GTPases constructs
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5391)
  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: 
            "unpublished"
COMMENT     Sequence Label: pBS L30 Flag-Apex2-Rab6B
FEATURES             Location/Qualifiers
     source          1..5391
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(31..53)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     153..172
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      239..694
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(326..345)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     536..557
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     820..842
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     834..851
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     835..851
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     858..877
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        858..876
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1289..1298
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1298..1321
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1322..2068
                     /codon_start=1
                     /product="soybean ascorbate peroxidase, engineered to serve
                     as a monomeric tag for electron microscopy and live-cell 
                     proteomics (Lam et al., 2015)"
                     /label=APEX2
                     /note="more active than the original APEX due to the A134P 
                     mutation"
                     /translation="GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGT
                     FDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAV
                     EVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHT
                     IGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDK
                     YAADEDAFFADYAEAHQKLSELGFADA"
     protein_bind    2102..2126
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2757..2781)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2825..2844)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2869..2917
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        complement(3133..3151)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3172..3188)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3172..3188)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3185..3207)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3196..3212
                     /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(3220..3226,3227..3244,3245..3250))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3265..3286
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3403..3420)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3574..4162)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3654..3673)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4333..5124,5125..5193))
                     /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     4956..4975
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5194..5298)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5366..5384
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
       61 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
      121 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
      181 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggac
      241 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
      301 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
      361 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
      421 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
      481 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
      541 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
      601 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
      661 gcgaatttta acaaaatatt aacgcttaca atttccattc gccattcagg ctgcgcaact
      721 gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat
      781 gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa
      841 cgacggccag tgaattgtaa tacgactcac tatagggcga attcgagctc ggtacccggg
      901 gatcgcagaa caaacgccca gaaccgaagg ccggagccca gacgccgcag cagcccagcc
      961 tgcgttgtaa cccggtcccc atagcaacca actaccgcag actactctcg caagcgctgt
     1021 tctgcttgtg acccggaccg gcttctcttc cgcttccggt cccacaatcc tctgtcggcc
     1081 tagaagagct ttgcattgtg ggagctcctt cctttctcgc tccccggcca tcttggcggc
     1141 tggtgttggt gagtgagctc tgcggggtaa acgattaggc ggctcgggga gctccgctag
     1201 ctggtgtttg acgctctgga ttataatcct ctcttctgtc ctctgtgtat gctaggttgg
     1261 gggttgtccc gcacctacta gaggatccgc caccatggac tacaaggatg acgacgataa
     1321 gggaaagtct tacccaactg tgagtgctga ttaccaggac gccgttgaga aggcgaagaa
     1381 gaagctcaga ggcttcatcg ctgagaagag atgcgctcct ctaatgctcc gtttggcatt
     1441 ccactctgct ggaacctttg acaagggcac gaagaccggt ggacccttcg gaaccatcaa
     1501 gcaccctgcc gaactggctc acagcgctaa caacggtctt gacatcgctg ttaggctttt
     1561 ggagccactc aaggcggagt tccctatttt gagctacgcc gatttctacc agttggctgg
     1621 cgttgttgcc gttgaggtca cgggtggacc taaggttcca ttccaccctg gaagagagga
     1681 caagcctgag ccaccaccag agggtcgctt gcccgatccc actaagggtt ctgaccattt
     1741 gagagatgtg tttggcaaag ctatggggct tactgaccaa gatatcgttg ctctatctgg
     1801 gggtcacact attggagctg cacacaagga gcgttctgga tttgagggtc cctggacctc
     1861 taatcctctt attttcgaca actcatactt cacggagttg ttgagtggtg agaaggaagg
     1921 tctccttcag ctaccttctg acaaggctct tttgtctgac cctgtattcc gccctctcgt
     1981 tgataaatat gcagcggacg aagatgcctt ctttgctgat tacgctgagg ctcaccaaaa
     2041 gctttccgag cttgggtttg ctgatgccgg tggttccggt ggatctggag gttcttacat
     2101 cacaagtttg tacaaaaaag caggcaccat gtccgcaggg ggagattttg ggaatccact
     2161 gagaaaattc aagttggtgt tcttggggga gcagagcgtc gggaagacgt ctctgattac
     2221 gaggttcatg tacgacagct tcgacaacac ataccaggca accattggga ttgacttctt
     2281 gtcaaaaacc atgtacttgg aggaccgcac ggtgcgactg cagctctggg acacagctgg
     2341 tcaggagagg ttccgcagcc tgatccccag ctacatccgg gactccacgg tggctgtggt
     2401 ggtgtacgac atcacaaatc tcaactcctt ccaacagacc tctaagtgga tcgacgacgt
     2461 caggacagag aggggcagtg atgttatcat catgctggtg ggcaacaaga cggacctggc
     2521 tgataagagg cagataacca tcgaggaggg ggagcagcgc gccaaagaac tgagcgtcat
     2581 gttcattgag accagtgcga agactggcta caacgtgaag cagctttttc gacgtgtggc
     2641 gtcggctcta cccggaatgg agaatgtcca ggagaaaagc aaagaaggga tgattgacat
     2701 caagctggac aaaccccagg agcccccggc cagcgagggc ggctgctcct gctagaaccc
     2761 agctttcttg tacaaagtgg tgatgtagta ggtcgacctg cagggggaga tgggggaggc
     2821 taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa
     2881 gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt tcggtcccag
     2941 ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct
     3001 tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc
     3061 ggggcggcaa gccctgccat agccacgggc cccgtgggtt agggacgggg tcccccatgc
     3121 aagcttttgt tccctttagt gagggttaat ttcgagcttg gcgtaatcat ggtcatagct
     3181 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     3241 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3301 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     3361 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3421 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3481 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3541 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     3601 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     3661 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     3721 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     3781 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     3841 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     3901 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     3961 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4021 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4081 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4141 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4201 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4261 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4321 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4381 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4441 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4501 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4561 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     4621 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     4681 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     4741 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     4801 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     4861 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     4921 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     4981 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5041 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5101 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5161 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5221 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5281 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     5341 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc g
//
