LOCUS       sequence-433021-        7578 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encoding dominant negative Rab5A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7578)
  AUTHORS   Jikko Y, Deguchi E, Matsuda K, Hino N, Tsukiji S, Matsuda M, Terai K
  TITLE     Front-biased activation of Ras-Rab5-Rac1 loop coordinates collective
            cell migration.
  JOURNAL   J Cell Sci. 2025 Jul 16:jcs.263779. doi: 10.1242/jcs.263779.
  PUBMED    40667649
REFERENCE   2  (bases 1 to 7578)
  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: "J Cell Sci.
            2025 Jul 16:jcs.263779. doi: 10.1242/jcs.263779."
COMMENT     Sequence Label: pPBpuro-Rab5S34N
FEATURES             Location/Qualifiers
     source          1..7578
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   674..708
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        1011..1390
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1392..1669
                     /label=chicken beta-actin promoter
     intron          1670..2686
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2610..2632
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2694..2713
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     misc_feature    3484..4033
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3628..3645)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3855..3874
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4036..4635
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4036..4055)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4532..4552
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4662..4679)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4668..4892
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4939..4955)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4963..4979
                     /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(4987..4993,4994..5011,5012..5017))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5032..5053
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     repeat_region   5287..5349
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5390..5410)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5390..5408)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5429..5445)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    5453..5469
                     /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(5477..5483,5484..5501,5502..5507))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5522..5543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5660..5677)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5831..6419)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5911..5930)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6590..7381,7382..7450))
                     /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     7213..7232
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7451..7555)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggggcgcgc
      661 cattctagat taaccctaga aagatagtct gcgtaaaatt gacgcatgca ttcttgaaat
      721 attgctctct ctttctaaat agcgcgaatc cgtcgctgtg catttaggac atctcagtcg
      781 ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta agtgtcactg attttgaact
      841 ataacgaccg cgtgagtcaa aatgacgcat gattatcttt tacgtgactt ttaagattta
      901 actcatacga taattatatt gttatttcat gttctactta cgtgataact tattatatat
      961 atattttctt gttatagata tcaactagaa tgctagcatg ggcccatctc gacattgatt
     1021 attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga
     1081 gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg
     1141 cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg
     1201 acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca
     1261 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
     1321 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
     1381 tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct cccccccctc
     1441 cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga tgggggcggg
     1501 gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag
     1561 gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc
     1621 gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc
     1681 gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg
     1741 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa
     1801 ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg
     1861 gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg
     1921 cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg
     1981 cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc
     2041 gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg
     2101 gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg
     2161 agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct
     2221 cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg
     2281 ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg
     2341 cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc
     2401 tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg
     2461 gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc
     2521 gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg
     2581 ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc
     2641 tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt
     2701 gctggttatt gtgctgtctc atcattttgg caaagaattg ggatccgaat tcctcgagat
     2761 ggctaatcga ggagcaacaa gacccaacgg gccaaatact ggaaataaaa tatgccagtt
     2821 caaactagta cttctgggag agtctgctgt tggcaaaaat agcctagtgc ttcgttttgt
     2881 gaagggccaa tttcatgaat ttcaagagag taccataggg gctgcttttc taacccaaac
     2941 tgtgtgtctt gatgatacaa cagtaaagtt tgaaatatgg gatacagctg gtcaagaacg
     3001 ataccatagc ttagcaccaa tgtactacag aggagcacaa gcagccatag ttgtatatga
     3061 tatcacaaat gaggagtcct ttgccagagc caaaaactgg gttaaagaac ttcagaggca
     3121 agccagtcct aacattgtaa tagctttatc aggaaacaag gctgatcttg caaataaaag
     3181 agctgtcgat ttccaggaag cacagtccta tgcagatgac aacagtttat tattcatgga
     3241 gacatcagct aaaacatcga tgaatgtaaa tgaaatattc atggcaatag ctaaaaagtt
     3301 gccaaagaac gaaccacaga atccaggagc aaattctgcc agaggaagag gagtagacct
     3361 tactgaaccc acgcagccaa ccaggagtca gtgttgtagt aactaagcgg ccgctctaga
     3421 gtcaactaac ttaagctagc aacggtttcc ctctagcggg atcaattccg cccccccccc
     3481 ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc tatatgttat
     3541 tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc cctgtcttct
     3601 tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg
     3661 tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct gtagcgaccc
     3721 tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa aagccacgtg
     3781 tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt tggatagttg
     3841 tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag gatgcccaga
     3901 aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt acatgtgttt
     3961 agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa
     4021 aaacacgata ataccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac
     4081 gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac
     4141 accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg
     4201 cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc
     4261 tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg
     4321 gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg
     4381 caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag
     4441 ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg
     4501 gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc
     4561 ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc
     4621 aagcccggtg cctgatcgat gatctagagc tcgctgatca gcctcgactg tgccttctag
     4681 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     4741 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     4801 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     4861 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa cctgcagccc
     4921 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     4981 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5041 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5101 ccagcggatc ttaaaagttt tgttacttta tagaagaaat tttgagtttt tgtttttttt
     5161 taataaataa ataaacataa ataaattgtt tgttgaattt attattagta tgtaagtgta
     5221 aatataataa aacttaatat ctattcaaat taataaataa acctcgatat acagaccgat
     5281 aaaacacatg cgtcaatttt acgcatgatt atctttaacg tacgtcacaa tatgattatc
     5341 tttctagggt taatctagta tacgcgttta attaatccag cttttgttcc ctttagtgag
     5401 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5461 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5521 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5581 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5641 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5701 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5761 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5821 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     5881 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     5941 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6001 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6061 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6121 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6181 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6241 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6301 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6361 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6421 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6481 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6541 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6601 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6661 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6721 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6781 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6841 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     6901 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     6961 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7021 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7081 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7141 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7201 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7261 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7321 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7381 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7441 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7501 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7561 ttccccgaaa agtgccac
//
