LOCUS       sequence-254567-        6836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Improved redshifted FRET-readout of Stathmin/OP18 interaction with 
            alpha-tubulin.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6836)
  AUTHORS   Van Geel O, Cheung S, Gadella TWJ
  TITLE     Combining optogenetics with sensitive FRET imaging to monitor local 
            microtubule manipulations.
  JOURNAL   Sci Rep. 2020 Apr 7;10(1):6034. doi: 10.1038/s41598-020-62874-3.
  PUBMED    32265472
REFERENCE   2  (bases 1 to 6836)
  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: "Sci Rep."; 
            date: "2020-04-7"; volume: "10(1):6034. doi"; pages: " 
            10.1038/s41598-020-62874-3"
COMMENT     Sequence Label: ROPY (sYFP2-OP18-mScI-R125I)
FEATURES             Location/Qualifiers
     source          1..6836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     18..40
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     102..121
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        163..281
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     CDS             356..373
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             383..400
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      419..428
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(425..427,428..430,431..1141)
                     /codon_start=1
                     /product="monomeric optimized variant of EYFP, identical to
                     mVenus but with L68V (Kremers et al., 2006)"
                     /label=SYFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(470..491)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(731..750)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1078..1099
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1619..2311
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLIGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDEPYK"
     primer_bind     complement(1749..1767)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2017..2036
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(2408..2427)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2473..2528
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2527..2546)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      2616..2663
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     2676..3381
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           2764..3136
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(2793..3381)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(3390..3423)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     rep_origin      complement(3591..4178)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3670..3689)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4191..5006)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNGDRVFRLAQAQSRMNNGLVGA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     4914..4933
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_recomb     5249..6076
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             5249..5373
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(5411..6016)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        6144..6447
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6449..6651
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6602..6622
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
ORIGIN
        1 gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc
       61 taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc tggttattgt
      121 gctgtctcat cattttggca aagaattgga tcggaccgaa atcaccacaa ttcagcaaat
      181 tgtgaacatc atcacgttca tctttccctg gttgccaaag gcccattttc ctgtcagtaa
      241 cgagaaggtc gcgaattcag gcgcttttta gactggtcgt agggagacca caacggtttc
      301 cctctagaaa taattttgtt taactataag aaggagatat accatgcggg gttctcatca
      361 tcaccaccat cacagcagcg gcctggtgcc gcgcggcagc tcgctagcgc taccggtcgc
      421 caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
      481 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
      541 ctacggcaag ctgaccctga agctgatctg caccaccggc aagctgcccg tgccctggcc
      601 caccctcgtg accaccctgg gctacggcgt gcagtgcttc gcccgctacc ccgaccacat
      661 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
      721 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
      781 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
      841 gcacaagctg gagtacaact acaacagcca caacgtctat atcaccgccg acaagcagaa
      901 gaacggcatc aaggccaact tcaagatccg ccacaacatc gaggacggcg gcgtgcagct
      961 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     1021 ccactacctg agctaccagt ccaagctgag caaagacccc aacgagaagc gcgatcacat
     1081 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     1141 gtccggactc agatctacga tggcatcttc tgatattcag gtgaaagagc tggagaagcg
     1201 cgcttcaggc caggcttttg agctgattct cagccctcgg tcaaaagaat ctgtccccga
     1261 tttccccctt tcccccccaa agaagaagga cctttccctg gaggaaatcc agaagaaatt
     1321 agaagctgca gaagaaagac gcaagtctca tgaggcggaa gtcttgaagc agctcgcgga
     1381 gaagcgggag catgagaagg aggtgctcca gaaagccatc gaggagaaca acaacttcag
     1441 caagatggcg gaggagaagc tgacccacaa aatggaggct aacaaagaga accgggaggc
     1501 gcagatggcg gccaagctgg agcgcttgcg agagaaggac aagcacgtgg aagaggtgcg
     1561 gaagaacaaa gaatccaaag accccgcgga tgagactgag gctgacggat ccggttcagt
     1621 gagcaagggc gaggcagtga tcaaggagtt catgcggttc aaggtgcaca tggagggctc
     1681 catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac
     1741 ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttctcct gggacatcct
     1801 gtcccctcag ttcatgtacg gctccagggc cttcatcaag caccccgccg acatccccga
     1861 ctactataag cagtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga
     1921 cggcggcgcc gtgaccgtga cccaggacac ctccctggag gacggcaccc tgatctacaa
     1981 ggtgaagctc atcggcacca acttccctcc tgacggcccc gtaatgcaga agaagacaat
     2041 gggctgggaa gcgtccaccg agcggttgta ccccgaggac ggcgtgctga agggcgacat
     2101 taagatggcc ctgcgcctga aggacggcgg ccgctacctg gcggacttca agaccaccta
     2161 caaggccaag aagcccgtgc agatgcccgg cgcctacaac gtcgaccgca agttggacat
     2221 cacctcccac aacgaggact acaccgtggt ggaacagtac gaacgctccg agggccgcca
     2281 ctccaccggc ggcatggacg agccgtacaa atgagcggcc gtgggccgcc ctcaggtgca
     2341 ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg
     2401 agatcgatct ttttccctct gccaaaaatt atggggacat catgaagccc cttgagcatc
     2461 tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg aattttttgt
     2521 gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag aatgagtatt
     2581 tggtttagag tttggcaaca tatgcccata tgtaactagc ataacccctt ggggcctcta
     2641 aacgggtctt gaggggtttt ttgctgaaag catgcggagg aaattctcct tgaagtttcc
     2701 ctggtgttca aagtaaagga gtttgcacca gacgcacctc tgttcactgg tccggcgtat
     2761 taaaacacga tacattgtta ttagtacatt tattaagcgc tagattctgt gcgttgttga
     2821 tttacagaca attgttgtac gtattttaat aattcattaa atttataatc tttagggtgg
     2881 tatgttagag cgaaaatcaa atgattttca gcgtctttat atctgaattt aaatattaaa
     2941 tcctcaatag atttgtaaaa taggtttcga ttagtttcaa acaagggttg tttttccgaa
     3001 ccgatggctg gactatctaa tggattttcg ctcaacgcca caaaacttgc caaatcttgt
     3061 agcagcaatc tagctttgtc gatattcgtt tgtgttttgt tttgtaataa aggttcgacg
     3121 tcgttcaaaa tattatgcgc ttttgtattt ctttcatcac tgtcgttagt atacaattga
     3181 ctcgacgtaa acacgttaaa tagagcttgg acatatttaa catcgggcgt gttagcttta
     3241 ttaggccgat tatcgtcgtc gtcccaaccc tcgtcgttag aagttgcttc cgaagacgat
     3301 tttgccatag ccacacgacg cctattaatt gtgtcggcta acacgtccgc gatcaaattt
     3361 gtagttgagc tttttggaat tgcgatcgca taacttcgta tagcatacat tatacgaagt
     3421 tataagctcg gaacgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     3481 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     3541 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     3601 ctccgccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3661 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3721 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3781 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3841 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3901 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3961 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4021 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4081 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4141 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg ttagaaaaac
     4201 tcatcgagca tcaagtgaaa ctgcaattta ttcatatcag gattatcaat accatatttt
     4261 tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca
     4321 agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc
     4381 ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt
     4441 gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca gccattacgc
     4501 tcgtcatcaa aatcactcgc accaaccaaa ccgttattca ttcgtgattg cgcctgagcg
     4561 agacgaaata cgcgatcgcc gttaaaagga caattacaaa caggaatcga atgcaaccgg
     4621 cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat
     4681 acctggaatg ctgttttccc tgggatcgca gtggtgagta accatgcatc atcaggagta
     4741 cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt tagcctgacc
     4801 atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc
     4861 gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga
     4921 gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg cctcgagcaa
     4981 gacgtttccc gttgaatatg gctcatactc ttcctttttc aatattattg aagcatttat
     5041 cagggttatt gtctcatgtc cgcgcgtttc ctgcatcttt taatcaaatc ccaagatgtg
     5101 tataaacgcg ccggtatgta taggaagagg tttatactaa actgttacat tgcaaacgtg
     5161 gtttcgtgtg ccaagtgtga aaaccgatgt ttaatcaagg ctctgacgca tttctacaac
     5221 cacgactcca agtgtgtggg tgaagtcatg catcttttaa tcaaatccca agatgtgtat
     5281 aaaccaccaa actgccaaaa aatgaaaact gtcgacaagc tctgtccgtt tgctggcaac
     5341 tgcaagggtc tcaatcctat ttgtaattat tgaataataa aacaattata aatgtcaaat
     5401 ttgtttttta ttaacgatac aaaccaaacg caacaagaac atttgtagta ttatctataa
     5461 ttgaaaacgc gtagttataa tcgctgaggt aatatttaaa atcattttca aatgattcac
     5521 agttaatttg cgacaatata attttatttt cacataaact agacgccttg tcgtcttctt
     5581 cttcgtattc cttctctttt tcatttttct cttcataaaa attaacatag ttattatcgt
     5641 atccatatat gtatctatcg tatagagtaa attttttgtt gtcataaata tatatgtctt
     5701 ttttaatggg gtgtatagta ccgctgcgca tagtttttct gtaatttaca acagtgctat
     5761 tttctggtag ttcttcggag tgtgttgctt taattattaa atttatataa tcaatgaatt
     5821 tgggatcgtc ggttttatac aatatgttgc cggcatagta cgcagcttct tctagttcaa
     5881 ttacaccatt ttttagcagc accggattaa cataactttc caaaatgttg tacgaaccgt
     5941 taaacaaaaa cagttcacct cccttttcta tactattgtc tgcgagcagt tgtttgttgt
     6001 taaaaataac agccattgta atgagacgca caaactaata tcacaaactg gaaatgtcta
     6061 tcaatatata gttgctgatg gccggcctat taatagtaat caattacggg gtcattagtt
     6121 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
     6181 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
     6241 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
     6301 gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga cggtaaatgg
     6361 cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg gcagtacatc
     6421 tacgtattag tcatcgctat taccatgctg atgcggtttt ggcagtacac caatgggcgt
     6481 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
     6541 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc cgccccgttg
     6601 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagacg tcgtttagtg
     6661 aaccgtcaga tcactagatg ctttattgcg gtagtttatc acagttaaat tgctaacgcc
     6721 agtctcgaac ttaacgtgca gaagttggtc gtgaggcact gggcaggtaa gtatcgggcc
     6781 ctttgtgcgg ggggagcggc tcggggctgt ccgcgggggg acggctgcct tcgggg
//
