LOCUS       sequence-245335-        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"
     primer_bind     128..147
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     misc_recomb     463..1290
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             463..587
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(625..1230)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1358..1661
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1663..1865
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1816..1836
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2068..2090
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2152..2171
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2213..2331
                     /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             2406..2423
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2433..2450
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2469..2478
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2475..2477,2478..2480,2481..3191)
                     /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(2520..2541)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2781..2800)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3128..3149
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3669..4361
                     /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(3799..3817)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4067..4086
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     complement(4458..4477)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4523..4578
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4577..4596)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4666..4713
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4726..5431
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4814..5186
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4843..5431)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(5440..5473)
                     /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(5641..6228)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5720..5739)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6241..6836,1..220))
                     /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"
ORIGIN
        1 agtttagcct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca
       61 gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc
      121 cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc
      181 gcggcctcga gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt
      241 attgaagcat ttatcagggt tattgtctca tgtccgcgcg tttcctgcat cttttaatca
      301 aatcccaaga tgtgtataaa cgcgccggta tgtataggaa gaggtttata ctaaactgtt
      361 acattgcaaa cgtggtttcg tgtgccaagt gtgaaaaccg atgtttaatc aaggctctga
      421 cgcatttcta caaccacgac tccaagtgtg tgggtgaagt catgcatctt ttaatcaaat
      481 cccaagatgt gtataaacca ccaaactgcc aaaaaatgaa aactgtcgac aagctctgtc
      541 cgtttgctgg caactgcaag ggtctcaatc ctatttgtaa ttattgaata ataaaacaat
      601 tataaatgtc aaatttgttt tttattaacg atacaaacca aacgcaacaa gaacatttgt
      661 agtattatct ataattgaaa acgcgtagtt ataatcgctg aggtaatatt taaaatcatt
      721 ttcaaatgat tcacagttaa tttgcgacaa tataatttta ttttcacata aactagacgc
      781 cttgtcgtct tcttcttcgt attccttctc tttttcattt ttctcttcat aaaaattaac
      841 atagttatta tcgtatccat atatgtatct atcgtataga gtaaattttt tgttgtcata
      901 aatatatatg tcttttttaa tggggtgtat agtaccgctg cgcatagttt ttctgtaatt
      961 tacaacagtg ctattttctg gtagttcttc ggagtgtgtt gctttaatta ttaaatttat
     1021 ataatcaatg aatttgggat cgtcggtttt atacaatatg ttgccggcat agtacgcagc
     1081 ttcttctagt tcaattacac cattttttag cagcaccgga ttaacataac tttccaaaat
     1141 gttgtacgaa ccgttaaaca aaaacagttc acctcccttt tctatactat tgtctgcgag
     1201 cagttgtttg ttgttaaaaa taacagccat tgtaatgaga cgcacaaact aatatcacaa
     1261 actggaaatg tctatcaata tatagttgct gatggccggc ctattaatag taatcaatta
     1321 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1381 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1441 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1501 ctgcccactt ggcagtacat caagtgtatc atatgccaag tccgccccct attgacgtca
     1561 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttacgg gactttccta
     1621 cttggcagta catctacgta ttagtcatcg ctattaccat gctgatgcgg ttttggcagt
     1681 acaccaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1741 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaata
     1801 accccgcccc gttgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1861 gacgtcgttt agtgaaccgt cagatcacta gatgctttat tgcggtagtt tatcacagtt
     1921 aaattgctaa cgccagtctc gaacttaacg tgcagaagtt ggtcgtgagg cactgggcag
     1981 gtaagtatcg ggccctttgt gcggggggag cggctcgggg ctgtccgcgg ggggacggct
     2041 gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta
     2101 gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg ggcaacgtgc
     2161 tggttattgt gctgtctcat cattttggca aagaattgga tcggaccgaa atcaccacaa
     2221 ttcagcaaat tgtgaacatc atcacgttca tctttccctg gttgccaaag gcccattttc
     2281 ctgtcagtaa cgagaaggtc gcgaattcag gcgcttttta gactggtcgt agggagacca
     2341 caacggtttc cctctagaaa taattttgtt taactataag aaggagatat accatgcggg
     2401 gttctcatca tcaccaccat cacagcagcg gcctggtgcc gcgcggcagc tcgctagcgc
     2461 taccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     2521 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     2581 gcgatgccac ctacggcaag ctgaccctga agctgatctg caccaccggc aagctgcccg
     2641 tgccctggcc caccctcgtg accaccctgg gctacggcgt gcagtgcttc gcccgctacc
     2701 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     2761 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     2821 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     2881 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcaccgccg
     2941 acaagcagaa gaacggcatc aaggccaact tcaagatccg ccacaacatc gaggacggcg
     3001 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     3061 tgcccgacaa ccactacctg agctaccagt ccaagctgag caaagacccc aacgagaagc
     3121 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     3181 agctgtacaa gtccggactc agatctacga tggcatcttc tgatattcag gtgaaagagc
     3241 tggagaagcg cgctgcaggc caggcttttg agctgattct cgcccctcgg tcaaaagaat
     3301 ctgtccccga tttccccctt gctcccccaa agaagaagga cctttccctg gaggaaatcc
     3361 agaagaaatt agaagctgca gaagaaagac gcaaggctca tgaggcggaa gtcttgaagc
     3421 agctcgcgga gaagcgggag catgagaagg aggtgctcca gaaagccatc gaggagaaca
     3481 acaacttcag caagatggcg gaggagaagc tgacccacaa aatggaggct aacaaagaga
     3541 accgggaggc gcagatggcg gccaagctgg agcgcttgcg agagaaggac aagcacgtgg
     3601 aagaggtgcg gaagaacaaa gaatccaaag accccgcgga tgagactgag gctgacggat
     3661 ccggttcagt gagcaagggc gaggcagtga tcaaggagtt catgcggttc aaggtgcaca
     3721 tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     3781 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttctcct
     3841 gggacatcct gtcccctcag ttcatgtacg gctccagggc cttcatcaag caccccgccg
     3901 acatccccga ctactataag cagtccttcc ccgagggctt caagtgggag cgcgtgatga
     3961 acttcgagga cggcggcgcc gtgaccgtga cccaggacac ctccctggag gacggcaccc
     4021 tgatctacaa ggtgaagctc atcggcacca acttccctcc tgacggcccc gtaatgcaga
     4081 agaagacaat gggctgggaa gcgtccaccg agcggttgta ccccgaggac ggcgtgctga
     4141 agggcgacat taagatggcc ctgcgcctga aggacggcgg ccgctacctg gcggacttca
     4201 agaccaccta caaggccaag aagcccgtgc agatgcccgg cgcctacaac gtcgaccgca
     4261 agttggacat cacctcccac aacgaggact acaccgtggt ggaacagtac gaacgctccg
     4321 agggccgcca ctccaccggc ggcatggacg agccgtacaa atgagcggcc gtgggccgcc
     4381 ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca
     4441 aataccactg agatcgatct ttttccctct gccaaaaatt atggggacat catgaagccc
     4501 cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg
     4561 aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag
     4621 aatgagtatt tggtttagag tttggcaaca tatgcccata tgtaactagc ataacccctt
     4681 ggggcctcta aacgggtctt gaggggtttt ttgctgaaag catgcggagg aaattctcct
     4741 tgaagtttcc ctggtgttca aagtaaagga gtttgcacca gacgcacctc tgttcactgg
     4801 tccggcgtat taaaacacga tacattgtta ttagtacatt tattaagcgc tagattctgt
     4861 gcgttgttga tttacagaca attgttgtac gtattttaat aattcattaa atttataatc
     4921 tttagggtgg tatgttagag cgaaaatcaa atgattttca gcgtctttat atctgaattt
     4981 aaatattaaa tcctcaatag atttgtaaaa taggtttcga ttagtttcaa acaagggttg
     5041 tttttccgaa ccgatggctg gactatctaa tggattttcg ctcaacgcca caaaacttgc
     5101 caaatcttgt agcagcaatc tagctttgtc gatattcgtt tgtgttttgt tttgtaataa
     5161 aggttcgacg tcgttcaaaa tattatgcgc ttttgtattt ctttcatcac tgtcgttagt
     5221 atacaattga ctcgacgtaa acacgttaaa tagagcttgg acatatttaa catcgggcgt
     5281 gttagcttta ttaggccgat tatcgtcgtc gtcccaaccc tcgtcgttag aagttgcttc
     5341 cgaagacgat tttgccatag ccacacgacg cctattaatt gtgtcggcta acacgtccgc
     5401 gatcaaattt gtagttgagc tttttggaat tgcgatcgca taacttcgta tagcatacat
     5461 tatacgaagt tataagctcg gaacgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5521 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5581 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5641 tttccatagg ctccgccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5701 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5761 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5821 gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5881 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5941 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6001 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6061 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6121 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6181 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6241 ttagaaaaac tcatcgagca tcaagtgaaa ctgcaattta ttcatatcag gattatcaat
     6301 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     6361 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     6421 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     6481 tgaatccggt gagaatggca aaagcttatg catttctttc cagacttgtt caacaggcca
     6541 gccattacgc tcgtcatcaa aatcactcgc accaaccaaa ccgttattca ttcgtgattg
     6601 cgcctgagcg agacgaaata cgcgatcgcc gttaaaagga caattacaaa caggaatcga
     6661 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     6721 ttcttctaat acctggaatg ctgttttccc tgggatcgca gtggtgagta accatgcatc
     6781 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagcc
//
