LOCUS       sequence-379351-        4774 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial isolation of mTurquoise2 with a T203Y mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4774)
  AUTHORS   van der Linden FH, Thornquist SC, ter Beek RM, Huijts JY, Hink MA, 
            Gadella Jr. TWJ, Maimon G, Goedhart J.
  TITLE     A green lifetime biosensor for calcium that remains bright over its 
            full dynamic range.
  JOURNAL   bioRxiv 2024.11.05.622032; doi: 
            https://doi.org/10.1101/2024.11.05.622032
REFERENCE   2  (bases 1 to 4774)
  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: "bioRxiv 
            2024.11.05.622032; doi: https://doi.org/10.1101/2024.11.05.622032"
COMMENT     Sequence Label: pFPO-His-mTurquoise2_T203Y
FEATURES             Location/Qualifiers
     source          1..4774
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(193..212)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    237..284
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      613..1201
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1102..1121
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        1349..1652
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1654..1856
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1807..1827
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2059..2081
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2143..2162
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        2204..2322
                     /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             2397..2414
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2424..2441
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2460..2469
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2466..2468,2469..2471,2472..3185)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2511..2532)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2772..2791)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3119..3140
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3278..3297)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3343..3398
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3397..3416)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3486..3533
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        3559..3663
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3688..3708)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3732..3942
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        3754..3950
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3801..3936
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             join(3985..4774,1..5)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             join(3985..4774,1..5)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4039..4058)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4649..4668
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg
       61 agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga
      121 cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccctag
      181 ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg ctatgacggc
      241 aataaaaaga cagaataaaa cgcacggtgt tgggtcgttt gttcataaac gcggggttcg
      301 gtcccagggc tggcactctg tcgatacccc accgagaccc cattggggcc aatacgcccg
      361 cgtttcttcc ttttccccac cccacccccc aagttcgggt gaaggcccag ggctcgcagc
      421 caacgtcggg gcggcaggcc ctgccatagc ctcaggttac tcatatatac tttagattga
      481 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
      541 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
      601 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
      661 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
      721 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
      781 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
      841 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
      901 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
      961 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     1021 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     1081 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     1141 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     1201 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     1261 gttctttcct gcgttatccc ctgaggccgg cctattaata gtaatcaatt acggggtcat
     1321 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     1381 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     1441 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     1501 tggcagtaca tcaagtgtat catatgccaa gtccgccccc tattgacgtc aatgacggta
     1561 aatggcccgc ctggcattat gcccagtaca tgaccttacg ggactttcct acttggcagt
     1621 acatctacgt attagtcatc gctattacca tgctgatgcg gttttggcag tacaccaatg
     1681 ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg
     1741 ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaat aaccccgccc
     1801 cgttgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agacgtcgtt
     1861 tagtgaaccg tcagatcact agatgcttta ttgcggtagt ttatcacagt taaattgcta
     1921 acgccagtct cgaacttaac gtgcagaagt tggtcgtgag gcactgggca ggtaagtatc
     1981 gggccctttg tgcgggggga gcggctcggg gctgtccgcg gggggacggc tgccttcggg
     2041 ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac cggcggctct agagcctctg
     2101 ctaaccatgt tcatgccttc ttctttttcc tacagctcct gggcaacgtg ctggttattg
     2161 tgctgtctca tcattttggc aaagaattgg atcggaccga aatcaccaca attcagcaaa
     2221 ttgtgaacat catcacgttc atctttccct ggttgccaaa ggcccatttt cctgtcagta
     2281 acgagaaggt cgcgaattca ggcgcttttt agactggtcg tagggagacc acaacggttt
     2341 ccctctagaa ataattttgt ttaactataa gaaggagata taccatgcgg ggttctcatc
     2401 atcaccacca tcacagcagc ggcctggtgc cgcgcggcag ctcgctagcg ctggatcccg
     2461 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     2521 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     2581 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2641 ccaccctcgt gaccaccctg tcctggggcg tgcagtgctt cgcccgctac cccgaccaca
     2701 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2761 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     2821 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2881 ggcacaagct ggagtacaac tactttagcg acaacgtcta tatcaccgcc gacaagcaga
     2941 agaacggcat caaggccaac ttcaagatcc gccacaacat cgaggacggc ggcgtgcagc
     3001 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     3061 accactacct gagctaccag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     3121 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     3181 agtaaaagct tgtgattaac ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt
     3241 ggccaatgcc ctggctcaca aataccactg agatcgatct ttttccctct gccaaaaatt
     3301 atggggacat catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt
     3361 tcattgcaat agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg
     3421 caaatcattt aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata
     3481 tgtaactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     3541 catgctcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3601 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3661 agtcctgagg cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3721 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
     3781 tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
     3841 cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc
     3901 tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa gatcgatcaa
     3961 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     4021 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     4081 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     4141 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
     4201 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     4261 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     4321 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4381 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4441 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4501 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4561 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4621 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4681 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4741 cgcatcgcct tctatcgcct tcttgacgag ttct
//
