LOCUS       sequence-190918-        5438 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Produces equimolar levels of two fluorescent proteins. It can be 
            used to measure practical brightness and as a negative control for 
            FRET.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5438)
  AUTHORS   Mastop M, Bindels DS, Shaner NC, Postma M, Gadella TWJ Jr, Goedhart 
            J
  TITLE     Characterization of a spectrally diverse set of fluorescent proteins
            as FRET acceptors for mTurquoise2.
  JOURNAL   Sci Rep. 2017 Sep 20;7(1):11999. doi: 10.1038/s41598-017-12212-x.
  PUBMED    28931898
REFERENCE   2  (bases 1 to 5438)
  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: "2017-09-20"; volume: "7(1):11999. doi"; pages: " 
            10.1038/s41598-017-12212-x"
COMMENT     Sequence Label: mKOkappa-2A-mTurquoise2
FEATURES             Location/Qualifiers
     source          1..5438
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      607..616
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             634..1281
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="SVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRVTM
                     AEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSASV
                     SAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMYLK
                     LEGGGNHKCQFKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             1309..1362
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     regulatory      1378..1387
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1384..1386,1387..1389,1390..2103)
                     /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(1429..1450)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1690..1709)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2037..2058
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2226..2347
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2263..2282)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2317..2336
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2354..2809)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2491..2512)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2703..2722
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2836..2940
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2942..3299
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2942..3133
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2965..2985)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3150..3285
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3334..4128
                     /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             3334..4128
                     /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(3388..3407)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3998..4017
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4316..4335)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4360..4407
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4736..5324
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5225..5244
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
      601 ccggtcgcca ccatggtgag caagggcgag gagagtgtga ttaaaccaga gatgaagatg
      661 aggtactaca tggacggctc cgtcaatggg catgagttca caattgaagg tgaaggcaca
      721 ggcagacctt acgagggaca tcaagagatg acactacgcg tcacaatggc cgagggcggg
      781 ccaatgcctt tcgcgtttga cttagtgtca cacgtgttct gttacggcca cagagtattt
      841 actaaatatc cagaagagat accagactat ttcaaacaag catttcctga aggcctgtca
      901 tgggaaaggt cgttggagtt cgaagatggt gggtccgctt cagtcagtgc gcatataagc
      961 cttagaggaa acaccttcta ccacaaatcc aaatttactg gggttaactt tcctgccgat
     1021 ggtcctatca tgcaaaacca aagtgttgat tgggagccat caaccgagaa aattactgcc
     1081 agcgacggag ttctgaaggg tgatgttacg atgtacctaa aacttgaagg aggcggcaat
     1141 cacaaatgcc aattcaagac tacttacaag gcggcaaaag agattcttga aatgccagga
     1201 gaccattaca tcggccatcg cctcgtcagg aaaaccgaag gcaacattac tgagcaggta
     1261 gaagatgcag tagctcattc cactggcgac gagctgtaca agtccggaga gggcagagga
     1321 agtcttctaa catgcggtga cgtggaggag aatcccggcc ctggggatcc accggtcgcc
     1381 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1441 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     1501 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1561 accctcgtga ccaccctgtc ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg
     1621 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1681 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1741 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1801 cacaagctgg agtacaacta ctttagcgac aacgtctata tcaccgccga caagcagaag
     1861 aacggcatca aggccaactt caagatccgc cacaacatcg aggacggcgg cgtgcagctc
     1921 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1981 cactacctga gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg
     2041 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2101 taaagcggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg
     2161 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     2221 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     2281 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     2341 tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     2401 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     2461 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     2521 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     2581 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     2641 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     2701 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     2761 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     2821 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     2881 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     2941 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     3001 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3061 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3121 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3181 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     3241 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     3301 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     3361 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     3421 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     3481 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     3541 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     3601 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     3661 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     3721 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     3781 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     3841 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     3901 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     3961 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4021 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4081 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4141 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4201 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4261 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     4321 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     4381 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     4441 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     4501 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     4561 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt
     4621 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     4681 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     4741 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4801 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4861 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     4921 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4981 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5041 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5101 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5161 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5221 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5281 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5341 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5401 cccctgattc tgtggataac cgtattaccg ccatgcat
//
