LOCUS       sequence-254570-        6836 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Unphosphorylatable mutant of Stathmin/OP18 FRET-sensor.
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-4A
FEATURES             Location/Qualifiers
     source          1..6836
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..304
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        306..508
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     459..479
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     711..733
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     795..814
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        856..974
                     /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             1049..1066
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1076..1093
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      1112..1121
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1118..1120,1121..1123,1124..1834)
                     /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(1163..1184)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1424..1443)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1771..1792
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2312..3004
                     /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(2442..2460)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2710..2729
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(3101..3120)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3166..3221
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3220..3239)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3309..3356
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3369..4074
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3457..3829
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3486..4074)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4083..4116)
                     /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(4284..4871)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4363..4382)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4884..5699)
                     /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     5607..5626
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_recomb     5942..6769
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             5942..6066
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6104..6709)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
ORIGIN
        1 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
       61 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      121 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      181 aagtccgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      241 catgacctta cgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      301 catgctgatg cggttttggc agtacaccaa tgggcgtgga tagcggtttg actcacgggg
      361 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      421 ggactttcca aaatgtcgta ataaccccgc cccgttgacg caaatgggcg gtaggcgtgt
      481 acggtgggag gtctatataa gcagacgtcg tttagtgaac cgtcagatca ctagatgctt
      541 tattgcggta gtttatcaca gttaaattgc taacgccagt ctcgaactta acgtgcagaa
      601 gttggtcgtg aggcactggg caggtaagta tcgggccctt tgtgcggggg gagcggctcg
      661 gggctgtccg cggggggacg gctgccttcg ggggggacgg ggcagggcgg ggttcggctt
      721 ctggcgtgtg accggcggct ctagagcctc tgctaaccat gttcatgcct tcttcttttt
      781 cctacagctc ctgggcaacg tgctggttat tgtgctgtct catcattttg gcaaagaatt
      841 ggatcggacc gaaatcacca caattcagca aattgtgaac atcatcacgt tcatctttcc
      901 ctggttgcca aaggcccatt ttcctgtcag taacgagaag gtcgcgaatt caggcgcttt
      961 ttagactggt cgtagggaga ccacaacggt ttccctctag aaataatttt gtttaactat
     1021 aagaaggaga tataccatgc ggggttctca tcatcaccac catcacagca gcggcctggt
     1081 gccgcgcggc agctcgctag cgctaccggt cgccaccatg gtgagcaagg gcgaggagct
     1141 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     1201 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagctgat
     1261 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgggctacgg
     1321 cgtgcagtgc ttcgcccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     1381 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     1441 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     1501 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     1561 ccacaacgtc tatatcaccg ccgacaagca gaagaacggc atcaaggcca acttcaagat
     1621 ccgccacaac atcgaggacg gcggcgtgca gctcgccgac cactaccagc agaacacccc
     1681 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagctacc agtccaagct
     1741 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     1801 cgggatcact ctcggcatgg acgagctgta caagtccgga ctcagatcta cgatggcatc
     1861 ttctgatatt caggtgaaag agctggagaa gcgcgctgca ggccaggctt ttgagctgat
     1921 tctcgcccct cggtcaaaag aatctgtccc cgatttcccc cttgctcccc caaagaagaa
     1981 ggacctttcc ctggaggaaa tccagaagaa attagaagct gcagaagaaa gacgcaaggc
     2041 tcatgaggcg gaagtcttga agcagctcgc ggagaagcgg gagcatgaga aggaggtgct
     2101 ccagaaagcc atcgaggaga acaacaactt cagcaagatg gcggaggaga agctgaccca
     2161 caaaatggag gctaacaaag agaaccggga ggcgcagatg gcggccaagc tggagcgctt
     2221 gcgagagaag gacaagcacg tggaagaggt gcggaagaac aaagaatcca aagaccccgc
     2281 ggatgagact gaggctgacg gatccggttc agtgagcaag ggcgaggcag tgatcaagga
     2341 gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac ggccacgagt tcgagatcga
     2401 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     2461 gggtggcccc ctgcccttct cctgggacat cctgtcccct cagttcatgt acggctccag
     2521 ggccttcatc aagcaccccg ccgacatccc cgactactat aagcagtcct tccccgaggg
     2581 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gccgtgaccg tgacccagga
     2641 cacctccctg gaggacggca ccctgatcta caaggtgaag ctcatcggca ccaacttccc
     2701 tcctgacggc cccgtaatgc agaagaagac aatgggctgg gaagcgtcca ccgagcggtt
     2761 gtaccccgag gacggcgtgc tgaagggcga cattaagatg gccctgcgcc tgaaggacgg
     2821 cggccgctac ctggcggact tcaagaccac ctacaaggcc aagaagcccg tgcagatgcc
     2881 cggcgcctac aacgtcgacc gcaagttgga catcacctcc cacaacgagg actacaccgt
     2941 ggtggaacag tacgaacgct ccgagggccg ccactccacc ggcggcatgg acgagccgta
     3001 caaatgagcg gccgtgggcc gccctcaggt gcaggctgcc tatcagaagg tggtggctgg
     3061 tgtggccaat gccctggctc acaaatacca ctgagatcga tctttttccc tctgccaaaa
     3121 attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta
     3181 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga
     3241 gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc
     3301 atatgtaact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     3361 aagcatgcgg aggaaattct ccttgaagtt tccctggtgt tcaaagtaaa ggagtttgca
     3421 ccagacgcac ctctgttcac tggtccggcg tattaaaaca cgatacattg ttattagtac
     3481 atttattaag cgctagattc tgtgcgttgt tgatttacag acaattgttg tacgtatttt
     3541 aataattcat taaatttata atctttaggg tggtatgtta gagcgaaaat caaatgattt
     3601 tcagcgtctt tatatctgaa tttaaatatt aaatcctcaa tagatttgta aaataggttt
     3661 cgattagttt caaacaaggg ttgtttttcc gaaccgatgg ctggactatc taatggattt
     3721 tcgctcaacg ccacaaaact tgccaaatct tgtagcagca atctagcttt gtcgatattc
     3781 gtttgtgttt tgttttgtaa taaaggttcg acgtcgttca aaatattatg cgcttttgta
     3841 tttctttcat cactgtcgtt agtatacaat tgactcgacg taaacacgtt aaatagagct
     3901 tggacatatt taacatcggg cgtgttagct ttattaggcc gattatcgtc gtcgtcccaa
     3961 ccctcgtcgt tagaagttgc ttccgaagac gattttgcca tagccacacg acgcctatta
     4021 attgtgtcgg ctaacacgtc cgcgatcaaa tttgtagttg agctttttgg aattgcgatc
     4081 gcataacttc gtatagcata cattatacga agttataagc tcggaacgct gcgctcggtc
     4141 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4201 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4261 aaaaaggccg cgttgctggc gtttttccat aggctccgcc ccctgacgag catcacaaaa
     4321 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4381 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4441 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4501 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4561 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4621 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4681 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4741 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4801 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4861 aaggatctca agaagatcct ttgttagaaa aactcatcga gcatcaagtg aaactgcaat
     4921 ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg taatgaagga
     4981 gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc tgcgattccg
     5041 actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag gttatcaagt
     5101 gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagctt atgcatttct
     5161 ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact cgcaccaacc
     5221 aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc gccgttaaaa
     5281 ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag cgcatcaaca
     5341 atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt ccctgggatc
     5401 gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat ggtcggaaga
     5461 ggcataaatt ccgtcagcca gtttagcctg accatctcat ctgtaacatc attggcaacg
     5521 ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata caatcgatag
     5581 attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata taaatcagca
     5641 tccatgttgg aatttaatcg cggcctcgag caagacgttt cccgttgaat atggctcata
     5701 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gtccgcgcgt
     5761 ttcctgcatc ttttaatcaa atcccaagat gtgtataaac gcgccggtat gtataggaag
     5821 aggtttatac taaactgtta cattgcaaac gtggtttcgt gtgccaagtg tgaaaaccga
     5881 tgtttaatca aggctctgac gcatttctac aaccacgact ccaagtgtgt gggtgaagtc
     5941 atgcatcttt taatcaaatc ccaagatgtg tataaaccac caaactgcca aaaaatgaaa
     6001 actgtcgaca agctctgtcc gtttgctggc aactgcaagg gtctcaatcc tatttgtaat
     6061 tattgaataa taaaacaatt ataaatgtca aatttgtttt ttattaacga tacaaaccaa
     6121 acgcaacaag aacatttgta gtattatcta taattgaaaa cgcgtagtta taatcgctga
     6181 ggtaatattt aaaatcattt tcaaatgatt cacagttaat ttgcgacaat ataattttat
     6241 tttcacataa actagacgcc ttgtcgtctt cttcttcgta ttccttctct ttttcatttt
     6301 tctcttcata aaaattaaca tagttattat cgtatccata tatgtatcta tcgtatagag
     6361 taaatttttt gttgtcataa atatatatgt cttttttaat ggggtgtata gtaccgctgc
     6421 gcatagtttt tctgtaattt acaacagtgc tattttctgg tagttcttcg gagtgtgttg
     6481 ctttaattat taaatttata taatcaatga atttgggatc gtcggtttta tacaatatgt
     6541 tgccggcata gtacgcagct tcttctagtt caattacacc attttttagc agcaccggat
     6601 taacataact ttccaaaatg ttgtacgaac cgttaaacaa aaacagttca cctccctttt
     6661 ctatactatt gtctgcgagc agttgtttgt tgttaaaaat aacagccatt gtaatgagac
     6721 gcacaaacta atatcacaaa ctggaaatgt ctatcaatat atagttgctg atggccggcc
     6781 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccg
//
