LOCUS       sequence-176986-        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(T17N)
FEATURES             Location/Qualifiers
     source          1..6677
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(14..33)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     260..280
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(289..717)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(399..420)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     611..630
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    763..987
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     976..993
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     promoter        1013..1031
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1013..1030
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(join(1059..1586,1587..1589,1590..1637))
                     /codon_start=1
                     /product="dominant negative mutant of human Rac family 
                     small GTPase 1"
                     /label=Rac1 (T17N)
                     /translation="MQAIKCVVVGDGAVGKNCLLISYTTNAFPGEYIPTVFDNYSANVM
                     VDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     CDS             complement(join(1644..2354,2355..2357,2358..2360))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1644..2354,2355..2357,2358..2360))
                     /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(1686..1707)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2035..2054
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2294..2315
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2357..2366
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2379..2398)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2380..2398)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2443..2646)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2472..2492)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2647..3026
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3198..3217
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3292..3396
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3397..3465,3466..4257)
                     /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     complement(3615..3634)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4428..5016
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4917..4936
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5170..5187
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5304..5325
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5340..5345,5346..5363,5364..5370)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5378..5394
                     /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)."
     primer_bind     5383..5405
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5402..5418
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5402..5418
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5467..5588
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5478..5497)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5532..5551
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(5762..6556)
                     /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(5873..5892)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     6483..6502
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(6623..6677,1..275))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6637..6677,1..95)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gaggcagagg cggcctcggc ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga
       61 gaatgggcgg aactgggcgg agttaggggc gggatgggcg gagttagggg cgggactatg
      121 gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga gcctggggac
      181 tttccacacc tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg
      241 gagcctgggg actttccaca ccctaactga cacacattcc acagaattaa ttcgcgttaa
      301 atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata
      361 aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac
      421 tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc
      481 cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa
      541 atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg
      601 cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg
      661 tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtggg
      721 gataccccct agagccccag ctggttcttt ccgcctcaga agccatagag cccaccgcat
      781 ccccagcatg cctgctattg tcttcccaat cctccccctt gctgtcctgc cccaccccac
      841 cccccagaat agaatgacac ctactcagac aatgcgatgc aatttcctca ttttattagg
      901 aaaggacagt gggagtggca ccttccaggg tcaaggaagg cacgggggag gggcaaacaa
      961 cagatggctg gcaactagaa ggcacagtcg aggctgatca gcgagctcta gcatttaggt
     1021 gacactatag aatagggccc tctagatgca tgctcgagtt acaacagcag gcattttctc
     1081 ttcctcttct tcacgggagg cgggcagagg actgctcgga tcgcttcgtc aaacactgtc
     1141 ttgaggcctc gctgtgtgag cgccgagcac tccaggtatt ttacagcacc aatctcctta
     1201 gccatggcta gaccctgcgg ataggtgatg ggagtcagct tcttctcctt cagtttctcg
     1261 atcgtgtctt tatcatccct aagatcaagt ttagttccca ctaggatgat gggagtgttg
     1321 ggacagtggt gccgcacctc aggataccac tttgcacgga cattttcaaa tgatgcagga
     1381 ctcacaaggg aaaagcaaat taagaacaca tctgtttgcg gataggatag ggggcgtaat
     1441 ctgtcataat cttcttgtcc agctgtatcc cataagccca gattcaccgg ttttccatct
     1501 accataacat tggcagaata attgtcaaag acagtaggga tatattctcc aggaaatgca
     1561 ttggttgtgt aactgatcag taggcagttt ttacctacag ctccgtctcc caccaccaca
     1621 cacttgatgg cctgcatgaa ttccttgtac agctcgtcca tgccgagagt gatcccggcg
     1681 gcggtcacga actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagg
     1741 gcggactggg tgctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg
     1801 gtgttctgct ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc
     1861 ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg
     1921 ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc
     1981 ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg
     2041 tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg
     2101 gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg
     2161 taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg
     2221 aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac
     2281 ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc
     2341 tcctcgccct tgctcaccat ggtggcaagc ttgggtctcc ctatagtgag tcgtattaat
     2401 ttcgataagc cagtaagcag tgggttctct agttagccag agagctctgc ttatatagac
     2461 ctcccaccgt acacgcctac cgcccatttg cgtcaatggg gcggagttgt tacgacattt
     2521 tggaaagtcc cgttgatttt ggtgccaaaa caaactccca ttgacgtcaa tggggtggag
     2581 acttggaaat ccccgtgagt caaaccgcta tccacgccca ttgatgtact gccaaaaccg
     2641 catcaccatg gtaatagcga tgactaatac gtagatgtac tgccaagtag gaaagtccca
     2701 taaggtcatg tactgggcat aatgccaggc gggccattta ccgtcattga cgtcaatagg
     2761 gggcgtactt ggcatatgat acacttgatg tactgccaag tgggcagttt accgtaaata
     2821 ctccacccat tgacgtcaat ggaaagtccc tattggcgtt actatgggaa catacgtcat
     2881 tattgacgtc aatgggcggg ggtcgttggg cggtcagcca ggcgggccat ttaccgtaag
     2941 ttatgtaacg cggaactcca tatatgggct atgaactaat gaccccgtaa ttgattacta
     3001 ttaataacta gtcaataatc aatgtcaacg cgtatatctg gcccgtacat cgcgaagcag
     3061 cgcaaaacgc ctaaccctaa gcagattctt catgcaattg tcggtcaagc cttgccttgt
     3121 tgtagcttaa attttgctcg cgcactactc agcgacctcc aacacacaag cagggagcag
     3181 atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc ataggggatc
     3241 gggagatctc ccgatccgtc gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc
     3301 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     3361 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     3421 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     3481 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     3541 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     3601 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     3661 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     3721 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     3781 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     3841 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     3901 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     3961 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     4021 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     4081 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     4141 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     4201 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     4261 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     4321 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     4381 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     4441 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     4501 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     4561 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     4621 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     4681 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     4741 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     4801 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     4861 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     4921 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     4981 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     5041 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     5101 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     5161 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
     5221 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     5281 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
     5341 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
     5401 acaggaaaca gctatgacca tgattacgcc aagctctagc tagaggtcga cggtatacag
     5461 acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag tgaaaaaaat
     5521 gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata agctgcaata
     5581 aacaagttgg ggtgggcgaa gaactccagc atgagatccc cgcgctggag gatcatccag
     5641 ccggcgtccc ggaaaacgat tccgaagccc aacctttcat agaaggcggc ggtggaatcg
     5701 aaatctcgtg atggcaggtt gggcgtcgct tggtcggtca tttcgaaccc cagagtcccg
     5761 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     5821 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
     5881 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     5941 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca
     6001 cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg
     6061 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
     6121 tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
     6181 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
     6241 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
     6301 cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
     6361 gcgctgcctc gtcctgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
     6421 ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt
     6481 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
     6541 catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagat ccgaaaatgg
     6601 atatacaagc tcccgggagc tttttgcaaa agcctaggcc tccaaaaaag cctcctcact
     6661 acttctggaa tagctca
//
