LOCUS       sequence-324229-        6765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses RAB35 in mammalian cells with a GFP tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6765)
  AUTHORS   Bonet-Ponce L, Beilina A, Williamson CD, Lindberg E, Kluss JH, 
            Saez-Atienzar S, Landeck N, Kumaran R, Mamais A, Bleck CKE, Li Y, 
            Cookson MR
  TITLE     LRRK2 mediates tubulation and vesicle sorting from lysosomes.
  JOURNAL   Sci Adv. 2020 Nov 11;6(46). pii: 6/46/eabb2454. doi: 
            10.1126/sciadv.abb2454. Print 2020 Nov.
  PUBMED    33177079
REFERENCE   2  (bases 1 to 6765)
  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: "2020-11-11"; volume: "6(46). pii: 6/46/eabb2454. doi"; pages:
            " 10.1126/sciadv.abb2454. Print 2020 Nov"
COMMENT     Sequence Label: GFP-RAB35
FEATURES             Location/Qualifiers
     source          1..6765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(905..907,908..910,911..1621)
                     /codon_start=1
                     /product="Cycle 3 GFP (Crameri et al., 1996)"
                     /label=Cycle 3 GFP
                     /note="mammalian codon-optimized"
                     /translation="MASKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFSYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(938..966)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     1561..1581
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    1643..1667
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2300..2324)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2356..2373)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2362..2586
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2632..3060
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2719..2738)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2929..2950
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3069..3089)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3074..3403
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3254..3389
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3316..3335
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3470..4264
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3524..3543)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4134..4153
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4438..4559
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4475..4494)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4529..4548
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4608..4624)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4608..4624)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4621..4643)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4632..4648
                     /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(4656..4662,4663..4680,4681..4686))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4701..4722
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4839..4856)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5010..5598)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5090..5109)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5769..6560,6561..6629))
                     /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     6392..6411
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6630..6734)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga
      901 caccatggcc agcaaaggag aagaactttt cactggagtt gtcccaattc ttgttgaatt
      961 agatggtgat gttaatgggc acaaattttc tgtcagtgga gagggtgaag gtgatgctac
     1021 atacggaaag cttaccctta aatttatttg cactactgga aaactacctg ttccatggcc
     1081 aacacttgtc actactttct cttatggtgt tcaatgcttt tcccgttatc cggatcatat
     1141 gaaacggcat gactttttca agagtgccat gcccgaaggt tatgtacagg aacgcactat
     1201 atctttcaaa gatgacggga actacaagac gcgtgctgaa gtcaagtttg aaggtgatac
     1261 ccttgttaat cgtatcgagt taaaaggtat tgattttaaa gaagatggaa acattctcgg
     1321 acacaaactc gagtacaact ataactcaca caatgtatac atcacggcag acaaacaaaa
     1381 gaatggaatc aaagctaact tcaaaattcg ccacaacatt gaagatggat ccgttcaact
     1441 agcagaccat tatcaacaaa atactccaat tggcgatggc cctgtccttt taccagacaa
     1501 ccattacctg tcgacacaat ctgccctttc gaaagatccc aacgaaaagc gtgaccacat
     1561 ggtccttctt gagtttgtaa ctgctgctgg gattacacat ggcatggatg agctctacaa
     1621 aagcggttcc ggaccggatc aaacaagttt gtacaaaaaa gcaggctccg aattcgccct
     1681 tatggcccgg gactacgacc acctcttcaa gctgctcatc atcggcgaca gcggtgtggg
     1741 caagagcagt ttactgttgc gttttgcaga caacactttc tcaggcagct acatcaccac
     1801 gatcggagtg gatttcaaga tccggaccgt ggagatcaac ggggagaagg tgaagctgca
     1861 gatctgggac acagcggggc aggagcgctt ccgcaccatc acctccacgt attatcgggg
     1921 gacccacggg gtcattgtgg tttacgacgt caccagtgcc gagtcctttg tcaacgtcaa
     1981 gcggtggctt cacgaaatca accagaactg tgatgatgtg tgccgaatat tagtgggtaa
     2041 taagaatgac gaccctgagc ggaaggtggt ggagacggaa gatgcctaca aattcgccgg
     2101 gcagatgggc atccagttgt tcgagaccag cgccaaggag aatgtcaacg tggaagagat
     2161 gttcaactgc atcacggagc tggtcctccg agcaaagaaa gacaacctgg caaaacagca
     2221 gcagcaacaa cagaacgatg tggtgaagct cacgaagaac agtaaacgaa agaaacgctg
     2281 ctgctaaagg gcgaattcga cccagctttc ttgtacaaag tggtgataat taattaagat
     2341 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     2401 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     2461 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     2521 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     2581 ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct
     2641 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     2701 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     2761 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     2821 ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct
     2881 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     2941 tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta taagggattt
     3001 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3061 aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     3121 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     3181 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     3241 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     3301 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca
     3361 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg
     3421 tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca
     3481 agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg
     3541 ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg
     3601 cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc
     3661 agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt
     3721 cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc
     3781 atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca
     3841 tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc
     3901 acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg
     3961 gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct
     4021 cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc
     4081 tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc
     4141 tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta
     4201 cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt
     4261 ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga
     4321 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     4381 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     4441 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     4501 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     4561 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     4621 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4681 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4741 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4801 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4861 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4921 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4981 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     5041 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     5101 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     5161 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     5221 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     5281 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     5341 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     5401 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     5461 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     5521 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     5581 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     5641 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5701 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5761 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5821 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5881 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5941 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     6001 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     6061 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     6121 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     6181 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     6241 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     6301 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     6361 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     6421 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     6481 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     6541 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     6601 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6661 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6721 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtc
//
