LOCUS       sequence-333286-        5388 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Rab8A 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 5388)
  AUTHORS   Raphael Gaudin Lab
  TITLE     Generation of APEX2-fused Rab GTPases constructs
  JOURNAL   unpublished
REFERENCE   2  (bases 1 to 5388)
  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            "unpublished"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-Rab8A
FEATURES             Location/Qualifiers
     source          1..5388
                     /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(2754..2778)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2822..2841)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2866..2914
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        complement(3130..3148)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3169..3185)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3169..3185)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3182..3204)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3193..3209
                     /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(3217..3223,3224..3241,3242..3247))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3262..3283
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3400..3417)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3571..4159)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3651..3670)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4330..5121,5122..5190))
                     /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     4953..4972
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5191..5295)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5363..5381
                     /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 ggcgaagacc tacgattacc tgttcaagct
     2161 gctgctgatc ggggactcgg gggtggggaa gacctgtgtc ctgttccgct tctccgagga
     2221 cgccttcaac tccactttta tctccaccat aggaattgac tttaaaatta ggaccataga
     2281 gctcgatggc aagagaatta aactgcagat atgggacaca gccggtcagg aacggtttcg
     2341 gacgatcaca acggcctact acaggggtgc aatgggcatc atgctggtct acgacatcac
     2401 caacgagaag tccttcgaca acatccggaa ctggattcgc aacattgagg agcacgcctc
     2461 tgcagacgtc gaaaagatga tactcgggaa caagtgtgat gtgaatgaca agagacaagt
     2521 ttccaaggaa cggggagaaa agctggccct cgactatgga atcaagttca tggagaccag
     2581 cgcgaaggcc aacatcaatg tggaaaatgc atttttcact ctcgccagag atatcaaagc
     2641 aaaaatggac aaaaaattgg aaggcaacag cccccagggg agcaaccagg gagtcaaaat
     2701 cacaccggac cagcagaaga ggagcagctt tttccgatgt gttcttctgt agaacccagc
     2761 tttcttgtac aaagtggtga tgtagtaggt cgacctgcag ggggagatgg gggaggctaa
     2821 ctgaaacacg gaaggagaca ataccggaag gaacccgcgc tatgacggca ataaaaagac
     2881 agaataaaac gcacgggtgt tgggtcgttt gttcataaac gcggggttcg gtcccagggc
     2941 tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg cgtttcttcc
     3001 ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc caacgtcggg
     3061 gcggcaagcc ctgccatagc cacgggcccc gtgggttagg gacggggtcc cccatgcaag
     3121 cttttgttcc ctttagtgag ggttaatttc gagcttggcg taatcatggt catagctgtt
     3181 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     3241 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     3301 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     3361 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3421 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3481 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3541 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3601 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3661 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3721 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3781 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3841 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3901 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3961 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4021 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4081 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4141 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4201 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4261 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4321 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     4381 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     4441 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     4501 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     4561 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     4621 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     4681 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     4741 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     4801 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     4861 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     4921 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     4981 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5041 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5101 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5161 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5221 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5281 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     5341 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcg
//
