LOCUS       sequence-377513-        5852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative control for ratiometric comparison between green 
            fluorescent proteins and sensors.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5852)
  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 5852)
  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-mScarletI-antiFRET-P2A2-linker_noSacI
FEATURES             Location/Qualifiers
     source          1..5852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_recomb     194..1021
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             194..318
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(356..961)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1089..1392
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1394..1596
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1547..1567
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1799..1821
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1883..1902
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1944..2062
                     /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             2137..2154
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2164..2181
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     regulatory      2200..2209
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2206..2901
                     /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="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(2339..2357)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2607..2626
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3022..3078
                     /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(3205..3224)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3270..3325
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3324..3343)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      3413..3460
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     3473..4178
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           3561..3933
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(3590..4178)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    complement(4187..4220)
                     /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(4388..4975)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4467..4486)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4988..5803)
                     /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     5711..5730
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 atgtccgcgc gtttcctgca tcttttaatc aaatcccaag atgtgtataa acgcgccggt
       61 atgtatagga agaggtttat actaaactgt tacattgcaa acgtggtttc gtgtgccaag
      121 tgtgaaaacc gatgtttaat caaggctctg acgcatttct acaaccacga ctccaagtgt
      181 gtgggtgaag tcatgcatct tttaatcaaa tcccaagatg tgtataaacc accaaactgc
      241 caaaaaatga aaactgtcga caagctctgt ccgtttgctg gcaactgcaa gggtctcaat
      301 cctatttgta attattgaat aataaaacaa ttataaatgt caaatttgtt ttttattaac
      361 gatacaaacc aaacgcaaca agaacatttg tagtattatc tataattgaa aacgcgtagt
      421 tataatcgct gaggtaatat ttaaaatcat tttcaaatga ttcacagtta atttgcgaca
      481 atataatttt attttcacat aaactagacg ccttgtcgtc ttcttcttcg tattccttct
      541 ctttttcatt tttctcttca taaaaattaa catagttatt atcgtatcca tatatgtatc
      601 tatcgtatag agtaaatttt ttgttgtcat aaatatatat gtctttttta atggggtgta
      661 tagtaccgct gcgcatagtt tttctgtaat ttacaacagt gctattttct ggtagttctt
      721 cggagtgtgt tgctttaatt attaaattta tataatcaat gaatttggga tcgtcggttt
      781 tatacaatat gttgccggca tagtacgcag cttcttctag ttcaattaca ccatttttta
      841 gcagcaccgg attaacataa ctttccaaaa tgttgtacga accgttaaac aaaaacagtt
      901 cacctccctt ttctatacta ttgtctgcga gcagttgttt gttgttaaaa ataacagcca
      961 ttgtaatgag acgcacaaac taatatcaca aactggaaat gtctatcaat atatagttgc
     1021 tgatggccgg cctattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     1081 gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc
     1141 cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat
     1201 tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat
     1261 catatgccaa gtccgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat
     1321 gcccagtaca tgaccttacg ggactttcct acttggcagt acatctacgt attagtcatc
     1381 gctattacca tgctgatgcg gttttggcag tacaccaatg ggcgtggata gcggtttgac
     1441 tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt ttggcaccaa
     1501 aatcaacggg actttccaaa atgtcgtaat aaccccgccc cgttgacgca aatgggcggt
     1561 aggcgtgtac ggtgggaggt ctatataagc agacgtcgtt tagtgaaccg tcagatcact
     1621 agatgcttta ttgcggtagt ttatcacagt taaattgcta acgccagtct cgaacttaac
     1681 gtgcagaagt tggtcgtgag gcactgggca ggtaagtatc gggccctttg tgcgggggga
     1741 gcggctcggg gctgtccgcg gggggacggc tgccttcggg ggggacgggg cagggcgggg
     1801 ttcggcttct ggcgtgtgac cggcggctct agagcctctg ctaaccatgt tcatgccttc
     1861 ttctttttcc tacagctcct gggcaacgtg ctggttattg tgctgtctca tcattttggc
     1921 aaagaattgg atcggaccga aatcaccaca attcagcaaa ttgtgaacat catcacgttc
     1981 atctttccct ggttgccaaa ggcccatttt cctgtcagta acgagaaggt cgcgaattca
     2041 ggcgcttttt agactggtcg tagggagacc acaacggttt ccctctagaa ataattttgt
     2101 ttaactataa gaaggagata taccatgcgg ggttctcatc atcaccacca tcacagcagc
     2161 ggcctggtgc cgcgcggcag ctcgctagcg ctgccggtcg ccaccatggt gagcaagggc
     2221 gaggcagtga tcaaggagtt catgcggttc aaggtgcaca tggagggctc catgaacggc
     2281 cacgagttcg agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc
     2341 aagctgaagg tgaccaaggg tggccccctg cccttctcct gggacatcct gtcccctcag
     2401 ttcatgtacg gctccagggc cttcatcaag caccccgccg acatccccga ctactataag
     2461 cagtccttcc ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgcc
     2521 gtgaccgtga cccaggacac ctccctggag gacggcaccc tgatctacaa ggtgaagctc
     2581 cgcggcacca acttccctcc tgacggcccc gtaatgcaga agaagacaat gggctgggaa
     2641 gcgtccaccg agcggttgta ccccgaggac ggcgtgctga agggcgacat taagatggcc
     2701 ctgcgcctga aggacggcgg ccgctacctg gcggacttca agaccaccta caaggccaag
     2761 aagcccgtgc agatgcccgg cgcctacaac gtcgaccgca agttggacat cacctcccac
     2821 aacgaggact acaccgtggt ggaacagtac gaacgctccg agggccgcca ctccaccggc
     2881 ggcatggacg agctgtacaa gtcgaccaga tctctggccg aggcggcagc taaggaggcc
     2941 gcggcgaagg aggcagccgc gaaagaagca gccgcaaagg aagcggccgc caaggctgct
     3001 gcaagatctc tcggatcggg tgctactaac ttcagcctgc tgaagcaggc tggagacgtg
     3061 gaggagaacc ctggaccttc gctagcgcta ccggtgcctc aacatgatgt acaagtaagt
     3121 gattaacctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     3181 gctcacaaat accactgaga tcgatctttt tccctctgcc aaaaattatg gggacatcat
     3241 gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt
     3301 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa
     3361 acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgt aactagcata
     3421 accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaagcat gcggaggaaa
     3481 ttctccttga agtttccctg gtgttcaaag taaaggagtt tgcaccagac gcacctctgt
     3541 tcactggtcc ggcgtattaa aacacgatac attgttatta gtacatttat taagcgctag
     3601 attctgtgcg ttgttgattt acagacaatt gttgtacgta ttttaataat tcattaaatt
     3661 tataatcttt agggtggtat gttagagcga aaatcaaatg attttcagcg tctttatatc
     3721 tgaatttaaa tattaaatcc tcaatagatt tgtaaaatag gtttcgatta gtttcaaaca
     3781 agggttgttt ttccgaaccg atggctggac tatctaatgg attttcgctc aacgccacaa
     3841 aacttgccaa atcttgtagc agcaatctag ctttgtcgat attcgtttgt gttttgtttt
     3901 gtaataaagg ttcgacgtcg ttcaaaatat tatgcgcttt tgtatttctt tcatcactgt
     3961 cgttagtata caattgactc gacgtaaaca cgttaaatag agcttggaca tatttaacat
     4021 cgggcgtgtt agctttatta ggccgattat cgtcgtcgtc ccaaccctcg tcgttagaag
     4081 ttgcttccga agacgatttt gccatagcca cacgacgcct attaattgtg tcggctaaca
     4141 cgtccgcgat caaatttgta gttgagcttt ttggaattgc gatcgcataa cttcgtatag
     4201 catacattat acgaagttat aagctcggaa cgctgcgctc ggtcgttcgg ctgcggcgag
     4261 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4321 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4381 tggcgttttt ccataggctc cgccccctga cgagcatcac aaaaatcgac gctcaagtca
     4441 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4501 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4561 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4621 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4681 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4741 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4801 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4861 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4921 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4981 tcctttgtta gaaaaactca tcgagcatca agtgaaactg caatttattc atatcaggat
     5041 tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc
     5101 agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa
     5161 tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag
     5221 tgacgactga atccggtgag aatggcaaaa gcttatgcat ttctttccag acttgttcaa
     5281 caggccagcc attacgctcg tcatcaaaat cactcgcacc aaccaaaccg ttattcattc
     5341 gtgattgcgc ctgagcgaga cgaaatacgc gatcgccgtt aaaaggacaa ttacaaacag
     5401 gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat
     5461 caggatattc ttctaatacc tggaatgctg ttttccctgg gatcgcagtg gtgagtaacc
     5521 atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca
     5581 gccagtttag cctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt
     5641 tcagaaacaa ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt
     5701 gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta
     5761 atcgcggcct cgagcaagac gtttcccgtt gaatatggct catactcttc ctttttcaat
     5821 attattgaag catttatcag ggttattgtc tc
//
