LOCUS       sequence-372932-        9084 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 9084)
  AUTHORS   Tominaga T, Sahai E, Chardin P, McCormick F, Courtneidge SA, Alberts
            AS
  TITLE     Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase
            signaling.
  JOURNAL   Mol Cell. 2000 Jan . 5(1):13-25.
  PUBMED    10678165
REFERENCE   2  (bases 1 to 9084)
  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: "Mol Cell. 
            2000 Jan . 5(1):13-25."
COMMENT     Sequence Label: pEFmEGFP-mDia2
FEATURES             Location/Qualifiers
     source          1..9084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     559..579
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     regulatory      623..632
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4236..4630
                     /label=beta-globin poly(A)
                     /note="human beta-globin polyadenylation signal"
     primer_bind     complement(4941..4958)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4941..4957)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4950..4972)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5166..5185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5285..5307
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5345..5363)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5431..5535
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5536..5604,5605..6396)
                     /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(5754..5773)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6567..7155
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7056..7075
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7309..7326
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7443..7464
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7479..7484,7485..7502,7503..7509)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7517..7533
                     /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     7522..7544
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7541..7557
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7541..7557
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        7671..8849
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          7902..8840
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     8797..8817
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             8921..8938
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             8948..8977
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             join(8993..9084,1..616)
                     /codon_start=1
                     /gene="mgfp5"
                     /product="GFP with folding enhancement mutations"
                     /label=mgfp5
                     /note="suitable for expression in plants due to removal of 
                     a cryptic intron"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKAN
                     FKTRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITHGMDELY"
     primer_bind     complement(9020..9048)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
ORIGIN
        1 gggtgaaggt gatgcaacat acggaaaact tacccttaaa tttatttgca ctactggaaa
       61 actacctgtt ccatggccaa cacttgtcac tactctcact tatggtgttc aatgcttttc
      121 aagataccca gatcatatga agcggcacga cttcttcaag agcgccatgc ctgagggata
      181 cgtgcaggag aggaccatct tcttcaagga cgacgggaac tacaagacac gtgctgaagt
      241 caagtttgag ggagacaccc tcgtcaacag gatcgagctt aagggaatcg atttcaagga
      301 ggacggaaac atcctcggcc acaagttgga atacaactac aactcccaca acgtatacat
      361 catggccgac aagcaaaaga acggcatcaa agccaacttc aagacccgcc acaacatcga
      421 agacggcggc gtgcaactcg ctgatcatta tcaacaaaat actccaattg gcgatggccc
      481 tgtcctttta ccagacaacc attacctgtc cacacaatct gccctttcga aagatcccaa
      541 cgaaaagaga gaccacatgg tccttcttga gtttgtaaca gctgctggga ttacacatgg
      601 catggatgaa ctatacggat cagccaccat ggagaggcac cgggcgcgcg ctctcggccg
      661 ggacagcaag tcgtcgcgga ggaagggctt gcagtccgcg ccgcccgctg gcccctacga
      721 gcccggggag aagcgaccca agttgcattt aaatattaga acactgacag atgatatgct
      781 ggacaaattt gccagtataa gaattccagg gagcaagaaa gagagacctc cccttcccca
      841 cctgaagact gtgtctggga tcagtgacag ctcatcactg tcctcagaga caatggaaaa
      901 caacccaaag gcgctgccag agagtgaagt cttgaagctt tttgagaaga tgatggaaga
      961 tatgaattta aatgaagata aaaaggcacc attgcgggaa aaagacttcg gtatcaaaaa
     1021 agaaatggtg atgcagtaca ttaatactgc ttctaagaca ggaagtctta gaagtagccg
     1081 acagatctca cctcaggaat ttctccatga gctgaaaatg ggttacacag acgagagact
     1141 tttcacgtat ctggagtcac tccgagtatc attgaccagt catcctgtga gttgggtgca
     1201 aagctttgga cacgagggac tcggattatt gctggacatt ttggaaaaac taattaatgg
     1261 gcaaatccaa gaaaaagttg tgaagaagac tcagcacaaa gtcatccagt gtctgagagc
     1321 cctgatgaac acacagtatg gcttagaaag gattatgagt gacaagagga gtctttcctt
     1381 gttggcaaaa gccatggatc ccaggcagcc cgctatgatg gcagacgtgg tgaagcttct
     1441 gtctgcagtg tgcattgtcg gcgaggaaag catccttgaa gaagtgttag aagccttgac
     1501 ttcagctgga gaagaaagaa agattgacag atttttttcc attgtggaag gcctccggca
     1561 taactcagtg aacctgcaag ttgcttgtat gcagctcata aatgctctcg ttacatctcc
     1621 tgatgatttg gacttcaggc ttcacctcag aaatgaattt atgcgttgtg gattgaaaga
     1681 gatattgcca aacttaaagg gcattaagaa tgatggcctg gatatacaac ttaaagtctt
     1741 tgatgagcac aaagaagaag atttgagtga gtttttccat cgccttgaag acattagagc
     1801 tgaacttgat gaagcatctg atgtttacag catgttatgg gacacagtta aggaaactcg
     1861 agcagaggga cattttcttt ctattcttca gcatctcctg ctcattcgca atgatcgttt
     1921 tataagagag cagtatttca aattaattga tgagtgtgtg tcacagattg tattacatag
     1981 agatggaacg gaccctgact tcacatacag aaaaagacta gatttggact taagtcagtt
     2041 tgtagatgtt tgcatagatc aggccaaact agatgagtgg gaagagaaag catccgaaca
     2101 ttgcaagaaa tttgaaaaag agtgtactga ccaccaagaa acccaggctc aattgcagaa
     2161 aagagaggca aagattaatg agcttcaagc agagttacaa gcttttaaat cccagtttgg
     2221 tgccttgcca cctggtacaa aaattccttt gcaaccttca gtagaaggtg aagctggccc
     2281 ttcagccctt cctcctgccc caccagcact cagtggagga gtgccgcctc ccccaccgcc
     2341 ccctcctcca ccacccccac cactcccagg aatgccaatg ccatttggtg gccctgtacc
     2401 accaccacct cctctgggat tcctgggtgg gcaaagctct attccattaa acctgccatt
     2461 tggtttgaaa ccaaagaaag aatttaagcc tgaaatcagc atgagaagat tgaattggtt
     2521 aaagatcgga ccaaatgaaa tgtctgagaa ctgcttctgg atcaaagtaa atgaaaataa
     2581 gtatgaaaat agggatttgc tttgtaaact tgagaacact ttttgttgcc aagaaaaaga
     2641 gaaaaggaat acaaatgact ttgatgagaa gaaagttatt aagaagagaa tgaaggaact
     2701 taaatttcta gatcctaaaa ttgctcagaa cctttcaatc ttcctgagct ccttccgggt
     2761 gccatatgag aaaatcagga cgatgatatt ggaagtggat gaaacacagt tgtcagagtc
     2821 catgattcag aacttaataa agcaccttcc tgatgaggag cagttgaagt cattgtccca
     2881 gtttagaagt gactataaca gtttgtgtga gcctgagcag ttcgctgttg tgatgagcaa
     2941 tgtgaagaga ctccggccac ggctcagtgc tattctcttt aagcttcaat ttgaagagca
     3001 ggtgaacaac atcaaacctg acatcatggc tgtcagtact gcctgcgagg agatcaagaa
     3061 gagcaaaggc tttagcaagt tgctggaact tgtgttgcta atgggaaact acatgaatgc
     3121 tggctcccgg aatgctcaaa ccttcggatt tgaccttagc tctctctgta aactgaagga
     3181 tacaaaatct gcagatcaga aaaccacact cctccatttc ctggtagatg tatgtgaaga
     3241 aaagcatgct gacatccttc actttgtgga cgatttggca catttagaca aagctagcag
     3301 agtctctgta gaaatgctgg aaaagaacgt gaagcagatg ggaaggcagc ttcaacagct
     3361 tgagaagaat ttggaaacct ttccccctcc tgaggacttg catgacaagt ttgtgataaa
     3421 gatgtccagc ttcgttatca gtgcgaacga gcagtatgaa aaactctcca cactactggg
     3481 cagcatgaca caattgtacc agagtataat gggctactat gctgtcgaca tgaagaaggt
     3541 ttccgtggaa gagtttttta atgatctgaa caacttcaga acttcattta tgctagcatt
     3601 aaaggaaaac atcaaaaaac gagaagcagc agaaaaggag aaacgtgcca ggatagcgaa
     3661 agagcgagca gagaaagagc gacttgaacg ccagcaagag aaaaagcgct tactagaaat
     3721 gaaaactgag ggagatgaga caggagtgat ggatagtctg ctggaggcct tgcagtcagg
     3781 ggctgccttc cgcgacagaa gaaaaaggac accaaagctg aaagatattc ggcagagtct
     3841 cagcccgatg tctcagaggc ctgttctcaa agtttgtaac catgaaaatc agaaaatgcc
     3901 gttgacagaa gggtcacgtc cacaccacag tatcaattgc aactccacca ggactccagt
     3961 cgccaaggag cttaattata atctagacac tcatgcgtct acagggagga tcaaggcagt
     4021 tgagaaggaa gcctgtaatg cagaaagcaa caaaaaaaag gaaatggaac ttcttggctc
     4081 tgttgctaaa agcgaatcag ttcctgaagt tgaagccctg ctggcaagat tacgagcttt
     4141 ataaactagt ctagaaattc accccaccag tgcaggctgc ctatcagaaa gtggtggctg
     4201 gtgtggctaa tgccctggcc cacaagtatc actaagctcg ctttcttgct gtccaatttc
     4261 tattaaaggt tcctttgttc cctaagtcca actactaaac tgggggatat tatgaagggc
     4321 cttgagcatc tggattctgc ctaataaaaa acatttattt tcattgcaat gatgtattta
     4381 aattatttct gaatatttta ctaaaaaggg aatgtgggag gtcagtgcat ttaaaacata
     4441 aagaaatgaa gagctagttc aaaccttggg aaaatacact atatcttaaa ctccatgaaa
     4501 gaaggtgagg ctgcaaacag ctaatgcaca ttggcaacag cccctgatgc ctatgcctta
     4561 ttcatccctc agaaaaggat tcaagtagag gcttgatttg gaggttaaag ttttgctatg
     4621 ctgtatttta cattacttat tgttttagct gtcctcatga atgtcttttc actacccatt
     4681 tgcttatcct gcatctctca gccttgactc cactcagttc tcttgcttag agataccacc
     4741 tttcccctga agtgttcctt ccatgtttta cggcgagatg gtttctcctc gcctggccac
     4801 tcagccttag ttgtctctgt tgtcttatag aggtctactt gaagaaggaa aaacaggggg
     4861 catggtttga ctgtcctgtg agcccttctt ccctgcctcc cccactcaca gtgacccgga
     4921 atctgcagcc caagcttggc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     4981 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     5041 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc
     5101 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact
     5161 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     5221 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     5281 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     5341 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     5401 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     5461 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     5521 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     5581 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     5641 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     5701 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     5761 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     5821 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     5881 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     5941 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     6001 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     6061 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     6121 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     6181 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     6241 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     6301 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     6361 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     6421 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     6481 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     6541 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     6601 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     6661 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     6721 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     6781 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     6841 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     6901 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     6961 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     7021 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     7081 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     7141 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     7201 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     7261 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     7321 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     7381 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     7441 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     7501 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg
     7561 attacgaatt aattcgagct cgcccagctt tgcaaagatg gataaagttt taaacagaga
     7621 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
     7681 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
     7741 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
     7801 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
     7861 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
     7921 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
     7981 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
     8041 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
     8101 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
     8161 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
     8221 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
     8281 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
     8341 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
     8401 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     8461 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     8521 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     8581 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     8641 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     8701 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     8761 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     8821 gtttttttct tccatttcag gtgtcgtgag gaattagctt gcttacattt gcttctgaca
     8881 caactgtgtt cactagcaac ctcaaacaga caccatgccg catcaccacc atcatcacgc
     8941 catgccggag cagaagctga tatccgagga ggacctggcc atggccggat ctagtaaagg
     9001 agaagaactt ttcactggag ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg
     9061 gcacaaattt tctgtcagtg gaga
//
