LOCUS       sequence-73927-e        5328 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expresses EGFP-Rab1AS25N (dominant negative mutant) in mammalian 
            cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5328)
  AUTHORS   Huang B, Hubber A, McDonough JA, Roy CR, Scidmore MA, Carlyon JA
  TITLE     The Anaplasma phagocytophilum-occupied vacuole selectively recruits 
            Rab-GTPases that are predominantly associated with recycling 
            endosomes.
  JOURNAL   Cell Microbiol. 2010 Sep 1;12(9):1292-307. doi: 
            10.1111/j.1462-5822.2010.01468.x. Epub 2010 Mar 25.
  PUBMED    20345488
REFERENCE   2  (bases 1 to 5328)
  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"; doi: 
            "10.1111/j.1462-5822.2010.01468.x"; journalName: "Cell Microbiol"; 
            date: "2010-09-1- 1"; volume: "12"; issue: "9"; pages: "1292-307"
COMMENT     Sequence Label: EGFP-Rab1AS25N
FEATURES             Location/Qualifiers
     source          1..5328
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    777..898
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(814..833)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     868..887
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(905..1360)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1042..1063)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1254..1273
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        1387..1491
                     /gene="bla"
                     /label=AmpR promoter
     promoter        1493..1850
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1493..1684
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1516..1536)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1701..1836
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             1885..2679
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             1885..2679
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1939..1958)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2549..2568
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(2867..2886)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2911..2958
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3287..3875
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3776..3795
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        4050..4353
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4354..4557
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4508..4528
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      4596..4605
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4602..4604,4605..4607,4608..5318)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4602..4604,4605..4607,4608..5318)
                     /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(4647..4668)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4908..4927)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5255..5276
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gatccaccat gtccagcatg aatcccgaat atgattattt attcaagtta cttctgattg
       61 gcgactcagg ggttggaaag aattgccttc ttcttaggtt tgcagatgat acatatacag
      121 aaagctacat cagcacaatt ggtgtggatt tcaaaataag aactatagag ttagacggga
      181 aaacaatcaa gcttcaaata tgggacacag caggccagga aagatttcga acaatcacct
      241 ccagttatta cagaggagcc catggcatca tagttgtgta tgatgtgaca gatcaggagt
      301 ccttcaataa tgttaaacag tggctgcagg aaatagatcg ttatgccagt gaaaatgtca
      361 acaaattgtt ggtagggaac aaatgtgatc tgaccacaaa gaaagtagta gactacacaa
      421 cagcgaagga atttgctgat tcccttggaa ttccgttttt ggaaaccagt gctaagaatg
      481 caacgaatgt agaacagtct ttcatgacga tggcagctga gattaaaaag cgaatgggtc
      541 ccggagcaac agctggtggt gctgagaagt ccaatgttaa aattcagagc actccagtca
      601 agcagtcagg tggaggttgc tgctaactcg acggtaccgc gggcccggga tccaccggat
      661 ctagataact gatcataatc agccatacca catttgtaga ggttttactt gctttaaaaa
      721 acctcccaca cctccccctg aacctgaaac ataaaatgaa tgcaattgtt gttgttaact
      781 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
      841 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttaac
      901 gcgtaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
      961 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     1021 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     1081 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     1141 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     1201 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     1261 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     1321 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga
     1381 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     1441 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tcctgaggcg
     1501 gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag
     1561 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt ggaaagtccc
     1621 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     1681 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     1741 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     1801 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa
     1861 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     1921 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     1981 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     2041 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
     2101 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     2161 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     2221 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     2281 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     2341 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     2401 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     2461 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     2521 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     2581 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     2641 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     2701 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     2761 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     2821 gggatctcat gctggagttc ttcgcccacc ctagggggag gctaactgaa acacggaagg
     2881 agacaatacc ggaaggaacc cgcgctatga cggcaataaa aagacagaat aaaacgcacg
     2941 gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata
     3001 ccccaccgag accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc
     3061 ccccaagttc gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca
     3121 tagcctcagg ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     3181 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     3241 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     3301 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     3361 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     3421 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     3481 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     3541 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     3601 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     3661 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     3721 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     3781 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     3841 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     3901 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
     3961 ctgtggataa ccgtattacc gccatgcatt agttattaat agtaatcaat tacggggtca
     4021 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
     4081 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
     4141 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
     4201 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
     4261 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
     4321 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
     4381 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
     4441 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
     4501 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctggt
     4561 ttagtgaacc gtcagatccg ctagcgctac cggtcgccac catggtgagc aagggcgagg
     4621 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     4681 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     4741 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     4801 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     4861 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     4921 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     4981 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     5041 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     5101 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     5161 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     5221 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     5281 ccgccgggat cactctcggc atggacgagc tgtacaagtc cggactca
//
