LOCUS       sequence-377512-        5873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative control for ratiometric comparison between red fluorescent 
            proteins and sensors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5873)
  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 5873)
  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: pDRESS-mTq2-antiFRET-P2A2-linker_noSacI
FEATURES             Location/Qualifiers
     source          1..5873
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1076..1379
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1381..1583
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1534..1554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1786..1808
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1870..1889
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1931..2049
                     /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             2124..2141
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2151..2168
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2187..2196
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2193..2195,2196..2198,2199..2909)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2238..2259)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2499..2518)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2846..2867
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3030..3086
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(3213..3232)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3278..3333
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3332..3351)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3421..3468
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3481..4186
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3569..3941
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3598..4186)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4195..4228)
                     /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(4396..4983)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4475..4494)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4996..5811)
                     /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     5719..5738
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tataggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttat acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggcct
     1021 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1081 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1141 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1201 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc
     1261 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1321 ccttacggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgc
     1381 tgatgcggtt ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc
     1441 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1501 ttccaaaatg tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt
     1561 gggaggtcta tataagcaga cgtcgtttag tgaaccgtca gatcactaga tgctttattg
     1621 cggtagttta tcacagttaa attgctaacg ccagtctcga acttaacgtg cagaagttgg
     1681 tcgtgaggca ctgggcaggt aagtatcggg ccctttgtgc ggggggagcg gctcggggct
     1741 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1801 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
     1861 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc
     1921 ggaccgaaat caccacaatt cagcaaattg tgaacatcat cacgttcatc tttccctggt
     1981 tgccaaaggc ccattttcct gtcagtaacg agaaggtcgc gaattcaggc gctttttaga
     2041 ctggtcgtag ggagaccaca acggtttccc tctagaaata attttgttta actataagaa
     2101 ggagatatac catgcggggt tctcatcatc accaccatca cagcagcggc ctggtgccgc
     2161 gcggcagctc gctagcgctg ccggtcgcca ccatggtgag caagggcgag gagctgttca
     2221 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     2281 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     2341 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgtcc tggggcgtgc
     2401 agtgcttcgc ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     2461 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     2521 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     2581 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac tttagcgaca
     2641 acgtctatat caccgccgac aagcagaaga acggcatcaa ggccaacttc aagatccgcc
     2701 acaacatcga ggacggcggc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2761 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc aagctgagca
     2821 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2881 tcactctcgg catggacgag ctgtacaagt cgaccagatc tctggccgag gcggcagcta
     2941 aggaggccgc ggcgaaggag gcagccgcga aagaagcagc cgcaaaggaa gcggccgcca
     3001 aggctgctgc aagatctctc ggatcgggtg ctactaactt cagcctgctg aagcaggctg
     3061 gagacgtgga ggagaaccct ggaccttcgc tagcgctacc ggtgcctcaa catgatgtac
     3121 aagtaagtga ttaacctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca
     3181 atgccctggc tcacaaatac cactgagatc gatctttttc cctctgccaa aaattatggg
     3241 gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt
     3301 gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat
     3361 catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgtaa
     3421 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaagcatgc
     3481 ggaggaaatt ctccttgaag tttccctggt gttcaaagta aaggagtttg caccagacgc
     3541 acctctgttc actggtccgg cgtattaaaa cacgatacat tgttattagt acatttatta
     3601 agcgctagat tctgtgcgtt gttgatttac agacaattgt tgtacgtatt ttaataattc
     3661 attaaattta taatctttag ggtggtatgt tagagcgaaa atcaaatgat tttcagcgtc
     3721 tttatatctg aatttaaata ttaaatcctc aatagatttg taaaataggt ttcgattagt
     3781 ttcaaacaag ggttgttttt ccgaaccgat ggctggacta tctaatggat tttcgctcaa
     3841 cgccacaaaa cttgccaaat cttgtagcag caatctagct ttgtcgatat tcgtttgtgt
     3901 tttgttttgt aataaaggtt cgacgtcgtt caaaatatta tgcgcttttg tatttctttc
     3961 atcactgtcg ttagtataca attgactcga cgtaaacacg ttaaatagag cttggacata
     4021 tttaacatcg ggcgtgttag ctttattagg ccgattatcg tcgtcgtccc aaccctcgtc
     4081 gttagaagtt gcttccgaag acgattttgc catagccaca cgacgcctat taattgtgtc
     4141 ggctaacacg tccgcgatca aatttgtagt tgagcttttt ggaattgcga tcgcataact
     4201 tcgtatagca tacattatac gaagttataa gctcggaacg ctgcgctcgg tcgttcggct
     4261 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     4321 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     4381 cgcgttgctg gcgtttttcc ataggctccg ccccctgacg agcatcacaa aaatcgacgc
     4441 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4501 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4561 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4621 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4681 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4741 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4801 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4861 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4921 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4981 caagaagatc ctttgttaga aaaactcatc gagcatcaag tgaaactgca atttattcat
     5041 atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag gagaaaactc
     5101 accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc cgactcgtcc
     5161 aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa gtgagaaatc
     5221 accatgagtg acgactgaat ccggtgagaa tggcaaaagc ttatgcattt ctttccagac
     5281 ttgttcaaca ggccagccat tacgctcgtc atcaaaatca ctcgcaccaa ccaaaccgtt
     5341 attcattcgt gattgcgcct gagcgagacg aaatacgcga tcgccgttaa aaggacaatt
     5401 acaaacagga atcgaatgca accggcgcag gaacactgcc agcgcatcaa caatattttc
     5461 acctgaatca ggatattctt ctaatacctg gaatgctgtt ttccctggga tcgcagtggt
     5521 gagtaaccat gcatcatcag gagtacggat aaaatgcttg atggtcggaa gaggcataaa
     5581 ttccgtcagc cagtttagcc tgaccatctc atctgtaaca tcattggcaa cgctaccttt
     5641 gccatgtttc agaaacaact ctggcgcatc gggcttccca tacaatcgat agattgtcgc
     5701 acctgattgc ccgacattat cgcgagccca tttataccca tataaatcag catccatgtt
     5761 ggaatttaat cgcggcctcg agcaagacgt ttcccgttga atatggctca tactcttcct
     5821 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgtccgcgc gtt
//
