LOCUS       sequence-268091-        5388 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Rab7A 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   Deffieu MS, Cesonyte I, Delalande F, Boncompain G, Dorobantu C, Song
            E, Lucansky V, Hirschler A, Cianferani S, Perez F, Carapito C, 
            Gaudin R
  TITLE     Rab7-harboring vesicles are carriers of the transferrin receptor 
            through the biosynthetic secretory pathway.
  JOURNAL   Sci Adv. 2021 Jan 8;7(2). pii: 7/2/eaba7803. doi: 
            10.1126/sciadv.aba7803. Print 2021 Jan.
  PUBMED    33523982
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: "Sci Adv."; 
            date: "2021-01-8"; volume: "7(2). pii: 7/2/eaba7803. doi"; pages: " 
            10.1126/sciadv.aba7803. Print 2021 Jan"
COMMENT     Sequence Label: pBS L30 Flag-Apex-Rab7A
FEATURES             Location/Qualifiers
     source          1..5388
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..43
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      455..464
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             464..487
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             488..1234
                     /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    1268..1292
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1920..1944)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1988..2007)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2032..2080
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     promoter        complement(2296..2314)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2335..2351)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2335..2351)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2348..2370)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2359..2375
                     /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(2383..2389,2390..2407,2408..2413))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2428..2449
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2566..2583)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2737..3325)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2817..2836)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3496..4287,4288..4356))
                     /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     4119..4138
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4357..4461)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4529..4547
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4585..4607)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4707..4726
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      4793..5248
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4880..4899)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5090..5111
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5374..5388,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(5388,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttgtaatacg actcactata gggcgaattc gagctcggta
       61 cccggggatc gcagaacaaa cgcccagaac cgaaggccgg agcccagacg ccgcagcagc
      121 ccagcctgcg ttgtaacccg gtccccatag caaccaacta ccgcagacta ctctcgcaag
      181 cgctgttctg cttgtgaccc ggaccggctt ctcttccgct tccggtccca caatcctctg
      241 tcggcctaga agagctttgc attgtgggag ctccttcctt tctcgctccc cggccatctt
      301 ggcggctggt gttggtgagt gagctctgcg gggtaaacga ttaggcggct cggggagctc
      361 cgctagctgg tgtttgacgc tctggattat aatcctctct tctgtcctct gtgtatgcta
      421 ggttgggggt tgtcccgcac ctactagagg atccgccacc atggactaca aggatgacga
      481 cgataaggga aagtcttacc caactgtgag tgctgattac caggacgccg ttgagaaggc
      541 gaagaagaag ctcagaggct tcatcgctga gaagagatgc gctcctctaa tgctccgttt
      601 ggcattccac tctgctggaa cctttgacaa gggcacgaag accggtggac ccttcggaac
      661 catcaagcac cctgccgaac tggctcacag cgctaacaac ggtcttgaca tcgctgttag
      721 gcttttggag ccactcaagg cggagttccc tattttgagc tacgccgatt tctaccagtt
      781 ggctggcgtt gttgccgttg aggtcacggg tggacctaag gttccattcc accctggaag
      841 agaggacaag cctgagccac caccagaggg tcgcttgccc gatcccacta agggttctga
      901 ccatttgaga gatgtgtttg gcaaagctat ggggcttact gaccaagata tcgttgctct
      961 atctgggggt cacactattg gagctgcaca caaggagcgt tctggatttg agggtccctg
     1021 gacctctaat cctcttattt tcgacaactc atacttcacg gagttgttga gtggtgagaa
     1081 ggaaggtctc cttcagctac cttctgacaa ggctcttttg tctgaccctg tattccgccc
     1141 tctcgttgat aaatatgcag cggacgaaga tgccttcttt gctgattacg ctgaggctca
     1201 ccaaaagctt tccgagcttg ggtttgctga tgccggtggt tccggtggat ctggaggttc
     1261 ttacatcaca agtttgtaca aaaaagcagg caccatgacc tctaggaaga aagtgttgct
     1321 gaaggttatc atcctgggag attctggagt cgggaagaca tcactcatga accagtatgt
     1381 gaataagaaa ttcagcaatc agtacaaagc cacaatagga gctgactttc tgaccaagga
     1441 ggtgatggtg gatgacaggc tagtcacaat gcagatatgg gacacagcag gacaggaacg
     1501 gttccagtct ctcggtgtgg ccttctacag aggtgcagac tgctgcgttc tggtatttga
     1561 tgtgactgcc cccaacacat tcaaaaccct agatagctgg agagatgagt ttctcatcca
     1621 ggccagtccc cgagatcctg aaaacttccc atttgttgtg ttgggaaaca agattgacct
     1681 cgaaaacaga caagtggcca caaagcgggc acaggcctgg tgctacagca aaaacaacat
     1741 tccctacttt gagaccagtg ccaaggaggc catcaacgtg gagcaggcgt tccagacgat
     1801 tgcacggaat gcacttaagc aggaaacgga ggtggagctg tacaacgaat ttcctgaacc
     1861 tatcaaactg gacaagaatg accgggccaa ggcctcggca gaaagctgca gttgctagaa
     1921 cccagctttc ttgtacaaag tggtgatgta gtaggtcgac ctgcaggggg agatggggga
     1981 ggctaactga aacacggaag gagacaatac cggaaggaac ccgcgctatg acggcaataa
     2041 aaagacagaa taaaacgcac gggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc
     2101 cagggctggc actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt
     2161 tcttcctttt ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac
     2221 gtcggggcgg caagccctgc catagccacg ggccccgtgg gttagggacg gggtccccca
     2281 tgcaagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat catggtcata
     2341 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     2401 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     2461 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     2521 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     2581 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2641 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2701 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2761 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2821 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2881 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2941 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3001 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3061 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3121 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3181 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3241 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3301 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3361 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3421 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3481 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3541 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3601 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3661 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3721 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3781 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3841 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3901 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3961 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4021 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4081 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4141 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4201 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4261 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4321 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4381 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4441 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     4501 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtctcgc
     4561 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     4621 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     4681 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     4741 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggacgcgccc
     4801 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     4861 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     4921 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     4981 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     5041 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5101 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     5161 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5221 tttaacaaaa tattaacgct tacaatttcc attcgccatt caggctgcgc aactgttggg
     5281 aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg
     5341 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgtt
//
