LOCUS       sequence-227748-        6674 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 6674)
  AUTHORS   Nalbant P, Hodgson L, Kraynov V, Toutchkine A, Hahn KM
  TITLE     Activation of endogenous Cdc42 visualized in living cells.
  JOURNAL   Science. 2004 Sep 10. 305(5690):1615-9.
  PUBMED    15361624
REFERENCE   2  (bases 1 to 6674)
  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. 
            2004 Sep 10. 305(5690):1615-9."
COMMENT     Sequence Label: pcDNA3-EGFP-Cdc42(T17N)
FEATURES             Location/Qualifiers
     source          1..6674
                     /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     629..648
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        629..647
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      661..670
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(667..669,670..672,673..1383)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(667..669,670..672,673..1383)
                     /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(712..733)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(973..992)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1320..1341
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(1390..1437,1438..1440,1441..1965)
                     /codon_start=1
                     /product="dominant negative mutant of human Rho family 
                     small GTPase Cdc42"
                     /label=Cdc42 (T17N)
                     /translation="MQTIKCVVVGDGAVGKNCLLISYTTNKFPSEYVPTVFDNYAVTVM
                     IGGEPYTLGLFDTAGQEDYDRLRPLSYPQTDVFLVCFSVVSPSSFENVKEKWVPEITHH
                     CPKTPFLLVGTQIDLRDDPSTIEKLAKNKQKPITPETAEKLARDLKAVKYVECSALTQK
                     GLKNVFDEAILAALEPPEPKKSRRCVLL"
     promoter        complement(1993..2011)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(1994..2011)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2031..2048)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2037..2261
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2307..2735
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2394..2413)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2604..2625
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2744..2764)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2749..3078
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2929..3064
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2991..3010
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3145..3939
                     /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(3199..3218)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3809..3828
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4113..4234
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4150..4169)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4204..4223
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4283..4299)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4283..4299)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4296..4318)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4307..4323
                     /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(4331..4337,4338..4355,4356..4361))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4376..4397
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4514..4531)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4685..5273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4765..4784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5444..6235,6236..6304))
                     /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     6067..6086
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6305..6409)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6484..6503)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
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 agctctctgg ctaactagag
      601 aacccactgc ttactggctt atcgaaatta atacgactca ctatagggag acccaagctt
      661 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      721 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      781 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
      841 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
      901 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      961 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1021 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1081 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1141 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1201 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1261 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1321 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1381 aaggaattca tgcagacaat taagtgtgtt gttgtgggcg atggtgctgt tggtaaaaat
     1441 tgtctcctga tatcctacac aacaaacaaa tttccatcgg aatatgtacc gactgttttt
     1501 gacaactatg cagtcacagt tatgattggt ggagagccat atactcttgg actttttgat
     1561 actgcagggc aagaggatta tgacagatta cgaccgctga gttatccaca aacagatgta
     1621 tttctagtct gtttttcagt ggtctctcca tcttcatttg aaaacgtgaa agaaaagtgg
     1681 gtgcctgaga taactcacca ctgtccaaag actcctttct tgcttgttgg gactcaaatt
     1741 gatctcagag atgacccctc tactattgag aaacttgcca agaacaaaca gaagcctatc
     1801 actccagaga ctgctgaaaa gctggcccgt gacctgaagg ctgtcaagta tgtggagtgt
     1861 tctgcactta cacagaaagg cctaaagaat gtatttgacg aagcaatatt ggctgccctg
     1921 gagcctccag aaccgaagaa gagccgcagg tgtgtgctgc tatgactcga gcatgcatct
     1981 agagggccct attctatagt gtcacctaaa tgctagagct cgctgatcag cctcgactgt
     2041 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2101 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2161 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2221 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     2281 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     2341 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2401 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2461 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2521 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2581 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2641 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2701 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     2761 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     2821 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2881 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     2941 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3001 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3061 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     3121 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3181 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3241 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3301 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     3361 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3421 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3481 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     3541 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     3601 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     3661 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     3721 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     3781 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     3841 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     3901 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     3961 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4021 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4081 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4141 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4201 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4261 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4321 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4381 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4441 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4501 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4561 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4621 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4681 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4741 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4801 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4861 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4921 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4981 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5041 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5101 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5161 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5221 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5281 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5341 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5401 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5461 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5521 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5581 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5641 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5701 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5761 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5821 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5881 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5941 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6001 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6061 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6121 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6181 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6241 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6301 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6361 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6421 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
     6481 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
     6541 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
     6601 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
     6661 cagatatacg cgtt
//
