LOCUS       sequence-180891-        6677 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6677)
  AUTHORS   Kraynov VS, Chamberlain C, Bokoch GM, Schwartz MA, Slabaugh S, Hahn 
            KM
  TITLE     Localized Rac activation dynamics visualized in living cells.
  JOURNAL   Science. 2000 Oct 13. 290(5490):333-7.
  PUBMED    11030651
REFERENCE   2  (bases 1 to 6677)
  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: "Science. 
            2000 Oct 13. 290(5490):333-7."
COMMENT     Sequence Label: pcDNA3-EGFP-Rac1(Q61L)
FEATURES             Location/Qualifiers
     source          1..6677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      142..277
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     204..223
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             358..1152
                     /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(412..431)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1022..1041
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1326..1447
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1363..1382)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1417..1436
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1496..1512)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1496..1512)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1509..1531)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1520..1536
                     /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(1544..1550,1551..1568,1569..1574))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1589..1610
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1727..1744)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1898..2486)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1978..1997)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2657..3448,3449..3517))
                     /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     3280..3299
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3518..3622)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3697..3716)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3888..4267
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4268..4471
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4422..4442
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4516..4535
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4516..4534
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4548..4557
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4554..4556,4557..4559,4560..5270)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4554..4556,4557..4559,4560..5270)
                     /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(4599..4620)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4860..4879)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5207..5228
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(5277..5456,5457..5459,5460..5855)
                     /codon_start=1
                     /product="constitutively active mutant of human Rac family 
                     small GTPase 1"
                     /label=Rac1 (Q61L)
                     /translation="MQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVM
                     VDGKPVNLGLWDTAGLEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     promoter        complement(5883..5901)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(5884..5901)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(5921..5938)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5927..6151
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      6197..6625
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6284..6303)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6494..6515
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(6634..6654)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        join(6639..6677,1..291)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
ORIGIN
        1 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
       61 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
      121 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
      181 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
      241 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
      301 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
      361 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
      421 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
      481 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
      541 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
      601 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
      661 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
      721 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
      781 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
      841 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
      901 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
      961 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     1021 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     1081 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     1141 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     1201 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     1261 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     1321 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     1381 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     1441 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     1501 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     1561 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     1621 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     1681 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     1741 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     1801 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     1861 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     1921 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     1981 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     2041 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     2101 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     2161 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     2221 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     2281 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     2341 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     2401 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     2461 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     2521 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     2581 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     2641 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     2701 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     2761 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     2821 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     2881 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     2941 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     3001 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     3061 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     3121 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     3181 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     3241 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     3301 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     3361 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     3421 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     3481 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     3541 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     3601 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat
     3661 cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata
     3721 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     3781 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     3841 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt
     3901 gactagttat taatagtaat caattacggg gtcattagtt catagcccat atatggagtt
     3961 ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc
     4021 attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg
     4081 tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat
     4141 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     4201 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     4261 taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg
     4321 gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca
     4381 acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg
     4441 tgtacggtgg gaggtctata taagcagagc tctctggcta actagagaac ccactgctta
     4501 ctggcttatc gaaattaata cgactcacta tagggagacc caagcttgcc accatggtga
     4561 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     4621 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     4681 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     4741 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     4801 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     4861 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     4921 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     4981 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     5041 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     5101 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     5161 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     5221 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag gaattcatgc
     5281 aggccatcaa gtgtgtggtg gtgggagacg gagctgtagg taaaacttgc ctactgatca
     5341 gttacacaac caatgcattt cctggagaat atatccctac tgtctttgac aattattctg
     5401 ccaatgttat ggtagatgga aaaccggtga atctgggctt atgggataca gctggactag
     5461 aagattatga cagattacgc cccctatcct atccgcaaac agatgtgttc ttaatttgct
     5521 tttcccttgt gagtcctgca tcatttgaaa atgtccgtgc aaagtggtat cctgaggtgc
     5581 ggcaccactg tcccaacact cccatcatcc tagtgggaac taaacttgat cttagggatg
     5641 ataaagacac gatcgagaaa ctgaaggaga agaagctgac tcccatcacc tatccgcagg
     5701 gtctagccat ggctaaggag attggtgctg taaaatacct ggagtgctcg gcgctcacac
     5761 agcgaggcct caagacagtg tttgacgaag cgatccgagc agtcctctgc ccgcctcccg
     5821 tgaagaagag gaagagaaaa tgcctgctgt tgtaactcga gcatgcatct agagggccct
     5881 attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt gccttctagt
     5941 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     6001 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     6061 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     6121 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     6181 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     6241 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     6301 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     6361 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     6421 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     6481 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     6541 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     6601 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     6661 agtccccagg ctcccca
//
