LOCUS       sequence-227751-        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(wt)
FEATURES             Location/Qualifiers
     source          1..6677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      13..22
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(19..21,22..24,25..735)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(19..21,22..24,25..735)
                     /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(64..85)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(325..344)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     672..693
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             742..1320
                     /codon_start=1
                     /product="human Rac family small GTPase 1"
                     /label=Rac1
                     /translation="MQAIKCVVVGDGAVGKTCLLISYTTNAFPGEYIPTVFDNYSANVM
                     VDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     promoter        complement(1348..1366)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1349..1366)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(1386..1403)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1392..1616
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1662..2090
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1749..1768)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1959..1980
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2099..2119)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2104..2433
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2284..2419
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2346..2365
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2500..3294
                     /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(2554..2573)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3164..3183
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3468..3589
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3505..3524)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3559..3578
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3638..3654)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3638..3654)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3651..3673)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3662..3678
                     /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(3686..3692,3693..3710,3711..3716))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3731..3752
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3869..3886)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4040..4628)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4120..4139)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4799..5590,5591..5659))
                     /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     5422..5441
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5660..5764)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5839..5858)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6030..6409
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6410..6613
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6564..6584
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6658..6677
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6658..6676
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 agacccaagc ttgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
       61 atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc
      121 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      181 cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc
      241 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      301 caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
      361 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
      421 ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg
      481 gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac
      541 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
      601 ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag
      661 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
      721 gacgagctgt acaaggaatt catgcaggcc atcaagtgtg tggtggtggg agacggagct
      781 gtaggtaaaa cttgcctact gatcagttac acaaccaatg catttcctgg agaatatatc
      841 cctactgtct ttgacaatta ttctgccaat gttatggtag atggaaaacc ggtgaatctg
      901 ggcttatggg atacagctgg acaagaagat tatgacagat tacgccccct atcctatccg
      961 caaacagatg tgttcttaat ttgcttttcc cttgtgagtc ctgcatcatt tgaaaatgtc
     1021 cgtgcaaagt ggtatcctga ggtgcggcac cactgtccca acactcccat catcctagtg
     1081 ggaactaaac ttgatcttag ggatgataaa gacacgatcg agaaactgaa ggagaagaag
     1141 ctgactccca tcacctatcc gcagggtcta gccatggcta aggagattgg tgctgtaaaa
     1201 tacctggagt gctcggcgct cacacagcga ggcctcaaga cagtgtttga cgaagcgatc
     1261 cgagcagtcc tctgcccgcc tcccgtgaag aagaggaaga gaaaatgcct gctgttgtaa
     1321 ctcgagcatg catctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg
     1381 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     1441 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     1501 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     1561 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     1621 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     1681 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     1741 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     1801 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     1861 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt
     1921 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg
     1981 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc
     2041 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg
     2101 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2161 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     2221 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     2281 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     2341 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga
     2401 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt
     2461 ttcggatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg
     2521 cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag
     2581 acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt
     2641 tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta
     2701 tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg
     2761 ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt
     2821 gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat
     2881 ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg
     2941 atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca
     3001 gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc
     3061 catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc
     3121 gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat
     3181 attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc
     3241 gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga
     3301 ctctggggtt cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt
     3361 ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga
     3421 tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg
     3481 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     3541 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta
     3601 taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     3661 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     3721 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     3781 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3841 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     3901 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     3961 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4021 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4081 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     4141 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     4201 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     4261 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     4321 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     4381 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     4441 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     4501 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     4561 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     4621 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     4681 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     4741 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     4801 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     4861 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     4921 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     4981 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5041 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     5101 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     5161 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     5221 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     5281 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     5341 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     5401 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     5461 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     5521 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     5581 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     5641 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     5701 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     5761 cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc
     5821 cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct
     5881 ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag
     5941 gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct
     6001 tcgcgatgta cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta
     6061 atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac
     6121 ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac
     6181 gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt
     6241 acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat
     6301 tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga
     6361 ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt
     6421 ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca
     6481 ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg
     6541 tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta
     6601 tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa
     6661 tacgactcac tataggg
//
