LOCUS       sequence-223318-        5274 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 5274)
  TITLE     Bokoch Lab GTPases
REFERENCE   2  (bases 1 to 5274)
  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"
COMMENT     Sequence Label: pRK5-myc-Rac1-T17N
FEATURES             Location/Qualifiers
     source          1..5274
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      912..921
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             921..950
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             join(963..1010,1011..1013,1014..1541)
                     /codon_start=1
                     /product="dominant negative mutant of human Rac family 
                     small GTPase 1"
                     /label=Rac1 (T17N)
                     /translation="MQAIKCVVVGDGAVGKNCLLISYTTNAFPGEYIPTVFDNYSANVM
                     VDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     CDS             963..1541
                     /codon_start=1
                     /product="human Rac family small GTPase 1"
                     /label=Rac1
                     /translation="MQAIKCVVVGDGAVGKNCLLISYTTNAFPGEYIPTVFDNYSANVM
                     VDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     regulatory      1561..1570
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1574..1708
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1611..1630)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1665..1684
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        1777..2134
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        1777..1968
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(1800..1820)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      1985..2120
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2154..2171)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2154..2170)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2163..2185)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2383..2838
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2470..2489)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2680..2701
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2855..2874)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2974..2996
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3034..3052)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3120..3224
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3225..3293,3294..4085)
                     /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(3443..3462)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4256..4844
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4745..4764
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4998..5015
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5132..5153
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5168..5173,5174..5191,5192..5198)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5206..5222
                     /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     5211..5233
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5230..5246
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5230..5246
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgattgaatt ggccaccatg gagcagaagc tgatctccga ggaggacctg ggatccggta
      961 ccatgcaggc catcaagtgt gtggtggtgg gagacggagc tgtaggtaaa aattgcctac
     1021 tgatcagtta cacaaccaat gcatttcctg gagaatatat ccctactgtc tttgacaatt
     1081 attctgccaa tgttatggta gatggaaaac cggtgaatct gggcttatgg gatacagctg
     1141 gacaagaaga ttatgacaga ttacgccccc tatcctatcc gcaaacagat gtgttcttaa
     1201 tttgcttttc ccttgtgagt cctgcatcat ttgaaaatgt ccgtgcaaag tggtatcctg
     1261 aggtgcggca ccactgtccc aacactccca tcatcctagt gggaactaaa cttgatctta
     1321 gggatgataa agacacgatc gagaaactga aggagaagaa gctgactccc atcacctatc
     1381 cgcagggtct agccatggct aaggagattg gtgccgtaaa atacctggag tgctcggcgc
     1441 tcacacagcg aggcctcaag acagtgtttg acgaagcgat ccgagcagtc ctctgcccgc
     1501 ctcccgtgaa gaagaggaag agaaaatgcc tgctgttgta agaattcctg cagaagcttg
     1561 gccgccatgg cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat
     1621 cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact
     1681 catcaatgta tcttatcatg tctggatcgg gaattaattc ggcgcagcac catggcctga
     1741 aataacctct gaaagaggaa cttggttagg taccttctga ggcggaaaga accagctgtg
     1801 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     1861 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     1921 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     1981 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     2041 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     2101 aggaggcttt tttggaggcc taggcttttg caaaaagctg ttaacagctt ggcactggcc
     2161 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     2221 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     2281 caacagttgc gcagcctgaa tggcgaatgg cgcctgatgc ggtattttct ccttacgcat
     2341 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     2401 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
     2461 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     2521 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     2581 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     2641 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     2701 gaacaacact caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     2761 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     2821 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     2881 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     2941 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3001 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     3061 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     3121 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3181 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     3241 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3301 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3361 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3421 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     3481 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3541 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3601 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3661 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3721 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     3781 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3841 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3901 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3961 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     4021 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     4081 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     4141 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4201 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4261 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4321 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4381 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4441 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4501 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4561 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4621 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4681 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4741 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4801 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4861 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4921 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4981 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     5041 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5101 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     5161 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     5221 aatttcacac aggaaacagc tatgacatga ttacgaatta attcgagctc gccc
//
