LOCUS       sequence-343817-        7150 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial co-expression of Drosophila Sep2 fused to monomeric EGFP 
            and of Drosophila Peanut.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7150)
  AUTHORS   Mavrakis M, Tsai FC, Koenderink GH
  TITLE     Purification of recombinant human and Drosophila septin hexamers for
            TIRF assays of actin-septin filament assembly.
  JOURNAL   Methods Cell Biol. 2016;136:199-220. doi: 
            10.1016/bs.mcb.2016.03.020. Epub 2016 Jun 14.
  PUBMED    27473911
REFERENCE   2  (bases 1 to 7150)
  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: "Methods 
            Cell Biol."; date: "2016"; volume: "136:199-220. doi"; pages: " 
            10.1016/bs.mcb.2016.03.020. Epub 2016 Jun 14"
COMMENT     Sequence Label: pnCS_mEGFP-DSep2_Peanut-Strep
FEATURES             Location/Qualifiers
     source          1..7150
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     81..100
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        81..99
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    100..124
                     /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)."
     RBS             139..161
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(169..171,172..174,175..885)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(214..235)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(475..494)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     822..843
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     RBS             2185..2207
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3832..3855
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     primer_bind     complement(3898..3916)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3912..3959
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4130..4921)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     promoter        complement(4922..5013)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5061..5799)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     CDS             complement(6009..7091)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7053..7072
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(7092..7150,1..19))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
       61 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
      121 ttccccacta gtaataattt tgtttaactt taagaaggag atatacatat ggtgagcaag
      181 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
      241 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
      301 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
      361 ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc
      421 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac
      481 ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
      541 gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac
      601 aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg
      661 aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag
      721 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc
      781 cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc
      841 gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagggtac cggcagcggt
      901 atgtccgttg aggtggattt cgtcgacaag aaggaggtgc acctccgcac cctgaagcag
      961 tccggtcacg tgggattcga cagcttgcct gaccaactgg tcaacaagag tgtccagaat
     1021 ggattcgtct tcaacgtcat gtgtataggc gaaaccggac tgggcaagtc gacgctgatg
     1081 gacacgctgt tcaacaccag cttcgagtcc acgccgagtc cccacacttt gccgagcgtc
     1141 aagttgaagg cacacaccta cgagctgcag gagagcaatg tgcgactcaa gctcaccatc
     1201 tgcgacacgg ttggctacgg cgatcagatc aacaaggacg actcgttcaa ggcggtggtg
     1261 gactacatcg acgcacagtt cgagaactat ctgcaggagg agctgaagat caagcgatcg
     1321 ctggtgacat gccacgacag ccgcatccac atctgcctct actttatttg ccccactggg
     1381 cacggcctca aatcgctgga cctggtgtgc atgaagaagc tggacagcaa ggtgaacata
     1441 attccggtga ttgccaaggc ggataccatt tccaaggttg agctacagcg cttcaaggcg
     1501 aagatcattc aggagctaaa cgccaatgga gtgcacatct accagttccc caccgacgat
     1561 gagacggtgg ccgagaccaa caccagcatg aactcgcaca ttcccttcgc cgtggtgggc
     1621 agcacggagt ttattaaggt gggcaacaag ctcatccgcg ctcgccagta cccctggggc
     1681 acggtgcagg tggagaacga gacgcactgc gactttgtga agctgcgcga gatgctcatt
     1741 cgcaccaaca tggaggatat gcgcgaaaag acacacacgc gccactacga gctgtatcgc
     1801 cagaagaggc tggagcaaat gggcttcagc gatgtggaca gcgacaacaa gcccatctcg
     1861 ttccagcaga cgttcgaggc aaagcgctcc aaccatttgg ccgagctgca gtccaaggag
     1921 gaggaggttc gtcagatgtt cgtgcagcgc gttaaggaga aggaggccga gctgaaggag
     1981 agcgagaagg acctgcacgc caagttcgag aagctaaagc gggatcacgc cgaggagaag
     2041 cgcaagctgg aagagtcgcg caaggcgctc gaggaggact accttgactt ccagcggcgc
     2101 aagcagcagt tggccaccgc ccaccacacg ctcaccctcg gcaagagcaa gaagaagtag
     2161 ggatcctaat agtctagtaa taattttgtt taactttaag aaggagatat acatatgaat
     2221 agtcctcgct cgaacgcggt taatggcggc agcggcggcg ccatctccgc tctgcccagc
     2281 actctggccc aactggctct gcgcgacaag cagcaggctg cgtcggcatc ggcgtcctct
     2341 gccacgaacg gcagcagcgg cagcgagtcc ttggtgggcg tcggtggacg accgcccaat
     2401 cagccgccca gtgtgccagt ggccgccagt ggcaagctgg acaccagcgg cggaggcgcc
     2461 tccaacggcg actcgaacaa actgacccac gatctgcagg agaaggagca ccagcaggcg
     2521 cagaagccac agaagccacc gctcccggtg cgccagaagc ccatggagat cgccggctat
     2581 gtgggcttcg ccaacctgcc caatcaggtc tatcgcaagg ccgtcaagcg gggattcgag
     2641 ttcactctga tggtggtggg cgccagtggg ctgggcaagt cgacgctgat caactccatg
     2701 ttcctgtcgg acatctacaa cgcggaacag tatccgggcc cctcgctgcg caaaaagaag
     2761 accgtggccg tggaggccac caaagtaatg ctcaaggaga acggcgtcaa tctgacactc
     2821 acggtggtgg acacacccgg attcggtgat gccgttgaca acagcaactg ctgggtgccc
     2881 atactggagt acgtggatag caagtacgag gagtacctga cagccgagtc gcgggtgtac
     2941 cgaaaaacca tttcggacag ccgggtgcac tgctgcctct acttcatagc gccatcggga
     3001 cacggactcc tgccgctgga cattgcctgc atgcagagcc tgtcggacaa ggtaaatctg
     3061 gtgccggtga tcgccaaggc ggacaccatg acgcccgacg aggtgcatct atttaagaag
     3121 cagatcctta acgagatcgc ccagcacaag atcaagatct acgacttccc cgccacgctg
     3181 gaggatgcgg ccgaggaggc caaaaccacg cagaatttgc gcagccgagt gccattcgcg
     3241 gtggtgggtg ccaacaccat catcgagcag gacggaaaga aggtccgggg taggcgttat
     3301 ccctggggcc tggtggaggt ggagaacctg acgcattgcg actttatagc gctgcgcaac
     3361 atggtcatac gcacgcacct tcaggacctc aaggacgtga cgaacaacgt ccactacgag
     3421 aactaccgct gccggaagct atccgagctg ggattggtgg acggcaaggc caggctgtcc
     3481 aacaagaacc cactcaccca gatggaggag gaaaagcggg agcacgagca gaagatgaag
     3541 aagatggagg ccgagatgga gcaggtgttt gacatgaagg tcaaggagaa gatgcagaag
     3601 ctcagggact cggagctgga gctggcgcgg cgccacgagg agcgcaagaa ggcgctggag
     3661 ctgcagatcc gggagctgga ggagaagaga cgcgagttcg agcgcgagaa gaaggagtgg
     3721 gaggatgtca accacgtgac gctcgaggag ctcaagcgac gcagcctggg ggccaacagc
     3781 agcacggaca atgtggatgg caagaaggag aagaaaaaga agggtctgtt ctggagccac
     3841 ccgcagttcg aaaaataagg atcctaatag tctagagcta gccctaggct gctgccaccg
     3901 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     3961 tgaaacctca ggcatttgag aagcacacgg tcacactgct tccggtagtc aataaaccgg
     4021 taaaccagca atagacataa gcggctattt aacgaccctg ccctgaaccg acgaccgggt
     4081 catcgtggcc ggatcttgcg gcccctcggc ttgaacgaat tgttagacat tatttgccga
     4141 ctaccttggt gatctcgcct ttcacgtagt ggacaaattc ttccaactga tctgcgcgcg
     4201 aggccaagcg atcttcttct tgtccaagat aagcctgtct agcttcaagt atgacgggct
     4261 gatactgggc cggcaggcgc tccattgccc agtcggcagc gacatccttc ggcgcgattt
     4321 tgccggttac tgcgctgtac caaatgcggg acaacgtaag cactacattt cgctcatcgc
     4381 cagcccagtc gggcggcgag ttccatagcg ttaaggtttc atttagcgcc tcaaatagat
     4441 cctgttcagg aaccggatca aagagttcct ccgccgctgg acctaccaag gcaacgctat
     4501 gttctcttgc ttttgtcagc aagatagcca gatcaatgtc gatcgtggct ggctcgaaga
     4561 tacctgcaag aatgtcattg cgctgccatt ctccaaattg cagttcgcgc ttagctggat
     4621 aacgccacgg aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg cggagaatct
     4681 cgctctctcc aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg
     4741 tttcatcaag ccttacggtc accgtaacca gcaaatcaat atcactgtgt ggcttcaggc
     4801 cgccatccac tgcggagccg tacaaatgta cggccagcaa cgtcggttcg agatggcgct
     4861 cgatgacgcc aactacctct gatagttgag tcgatacttc ggcgatcacc gcttccctca
     4921 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4981 acatatttga atgtatttag aaaaataaac aaatagctag gtcactcggt cgctacgctc
     5041 cgggcgtgag actgcggcgg gcgctgcgga cacatacaaa gttacccaca gattccgtgg
     5101 ataagcaggg gactaacatg tgaggcaaaa cagcagggcc gcgccggtgg cgtttttcca
     5161 taggctccgc cctcctgcca gagttcacat aaacagacgc ttttccggtg catctgtggg
     5221 agccgtgagg ctcaaccatg aatctgacag tacgggcgaa acccgacagg acttaaagat
     5281 ccccaccgtt tccggcgggt cgctccctct tgcgctctcc tgttccgacc ctgccgttta
     5341 ccggatacct gttccgcctt tctcccttac gggaagtgtg gcgctttctc atagctcaca
     5401 cactggtatc tcggctcggt gtaggtcgtt cgctccaagc tgggctgtaa gcaagaactc
     5461 cccgttcagc ccgactgctg cgccttatcc ggtaactgtt cacttgagtc caacccggaa
     5521 aagcacggta aaacgccact ggcagcagcc attggtaact gggagttcgc agaggatttg
     5581 tttagctaaa cacgcggttg ctcttgaagt gtgcgccaaa gtccggctac actggaagga
     5641 cagatttggt tgctgtgctc tgcgaaagcc agttaccacg gttaagcagt tccccaactg
     5701 acttaacctt cgatcaaacc acctccccag gtggtttttt cgtttacagg gcaaaagatt
     5761 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctactga accgctcttg
     5821 atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat acgatataag
     5881 ttgtaattct catgttagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     5941 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     6001 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     6061 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     6121 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     6181 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     6241 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca
     6301 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     6361 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     6421 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     6481 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     6541 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     6601 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     6661 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     6721 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     6781 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     6841 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     6901 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     6961 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     7021 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     7081 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     7141 cgaaaggttt
//
