LOCUS       sequence-191168-        5456 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 5456)
  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 5456)
  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: mNeonGreen-2A-mTurquoise2
FEATURES             Location/Qualifiers
     source          1..5456
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        59..250
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     207..227
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(252..356)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      383..838
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(470..489)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     680..701
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    845..966
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(856..875)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     910..929
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(1089..1802,1803..1805,1806..1808))
                     /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(1134..1155)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1483..1502
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1742..1763
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1805..1814
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(1830..1883)
                     /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"
     CDS             complement(1890..2597)
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      2333..2342
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      2594..2603
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(2642..2845)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2671..2691)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2846..3149
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     rep_origin      complement(3324..3912)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3404..3423)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4241..4288
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     4313..4332
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(4520..5314)
                     /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             complement(4520..5314)
                     /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(4631..4650)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     5241..5260
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(join(5349..5456,1..250))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(5363..5456,1..42)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 gggcggagaa tgggcggaac tgggcggagt taggggcggg atgggcggag ttaggggcgg
       61 gactatggtt gctgactaat tgagatgcat gctttgcata cttctgcctg ctggggagcc
      121 tggggacttt ccacacctgg ttgctgacta attgagatgc atgctttgca tacttctgcc
      181 tgctggggag cctggggact ttccacaccc taactgacac acattccaca gctggttctt
      241 tccgcctcag gactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
      301 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
      361 tttccccgaa aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
      421 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
      481 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
      541 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
      601 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
      661 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
      721 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
      781 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttac
      841 gccttaagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
      901 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
      961 caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag
     1021 gttttttaaa gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tctagagtcg
     1081 cggccgcttt acttgtacag ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac
     1141 tccagcagga ccatgtgatc gcgcttctcg ttggggtctt tgctcagctt ggactgggtg
     1201 ctcaggtagt ggttgtcggg cagcagcacg gggccgtcgc cgatgggggt gttctgctgg
     1261 tagtggtcgg cgagctgcac gccgccgtcc tcgatgttgt ggcggatctt gaagttggcc
     1321 ttgatgccgt tcttctgctt gtcggcggtg atatagacgt tgtcgctaaa gtagttgtac
     1381 tccagcttgt gccccaggat gttgccgtcc tccttgaagt cgatgccctt cagctcgatg
     1441 cggttcacca gggtgtcgcc ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg
     1501 tccttgaaga agatggtgcg ctcctggacg tagccttcgg gcatggcgga cttgaagaag
     1561 tcgtgctgct tcatgtggtc ggggtagcgg gcgaagcact gcacgcccca ggacagggtg
     1621 gtcacgaggg tgggccaggg cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc
     1681 agcttgccgt aggtggcatc gccctcgccc tcgccggaca cgctgaactt gtggccgttt
     1741 acgtcgccgt ccagctcgac caggatgggc accaccccgg tgaacagctc ctcgcccttg
     1801 ctcaccatgg tggcgaccgg tggatcccca gggccgggat tctcctccac gtcaccgcat
     1861 gttagaagac ttcctctgcc ctctccggac ttgtacagct cgtccatgcc catcacatcg
     1921 gtaaaggcct tttgccactc cttgaagttg agctcggtct tggagtgctt gagctccgtc
     1981 ttacggaaca cgtacatcgg ctggttcttc agatagttag ccgccattgg cttggcaaag
     2041 gtgtaggtgg tccgcgcagt gctccggtag cgcttgccat ttccagtggt gtaactccac
     2101 ttaaaggtac tgatgatggt tttgtcgttg gggtaagtct tcttcgacct gcaccagtcc
     2161 gcagcggtca gcgagttggt catcacagga ccgtcagcag ggaaaccagt ccccttcacc
     2221 tgggcctctc ctttgatgtg gcttccctcg taggtgtagc ggtagttaac agtaagggag
     2281 gcaccatctt caaactgcat tgtgcgatgg acttggtagc cggagccatc taccatggcg
     2341 gcctggaaag gcgacatccc gtcagggtag ggcaggtact gatggaagcc atacccgata
     2401 tgagggacca gaatccaggg ggagaactgg aggtcaccct tggtggactt caggtttaac
     2461 tcctcataac catcatttgg attgccggtg ccctgaccca ccatgtcaaa gtccacaccg
     2521 ttgatggagc caaagatgtg taactcatgt gtcgctggga gagaggccat gttatcctcc
     2581 tcgcccttgc tcaccatggt ggcgaccggt agcgctagcg gatctgacgg ttcactaaac
     2641 cagctctgct tatatagacc tcccaccgta cacgcctacc gcccatttgc gtcaatgggg
     2701 cggagttgtt acgacatttt ggaaagtccc gttgattttg gtgccaaaac aaactcccat
     2761 tgacgtcaat ggggtggaga cttggaaatc cccgtgagtc aaaccgctat ccacgcccat
     2821 tgatgtactg ccaaaaccgc atcaccatgg taatagcgat gactaatacg tagatgtact
     2881 gccaagtagg aaagtcccat aaggtcatgt actgggcata atgccaggcg ggccatttac
     2941 cgtcattgac gtcaataggg ggcgtacttg gcatatgata cacttgatgt actgccaagt
     3001 gggcagttta ccgtaaatac tccacccatt gacgtcaatg gaaagtccct attggcgtta
     3061 ctatgggaac atacgtcatt attgacgtca atgggcgggg gtcgttgggc ggtcagccag
     3121 gcgggccatt taccgtaagt tatgtaacgc ggaactccat atatgggcta tgaactaatg
     3181 accccgtaat tgattactat taataactaa tgcatggcgg taatacggtt atccacagaa
     3241 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     3301 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     3361 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     3421 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     3481 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     3541 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     3601 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     3661 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     3721 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     3781 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3841 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3901 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3961 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     4021 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaacc tgaggctatg
     4081 gcagggcctg ccgccccgac gttggctgcg agccctgggc cttcacccga acttgggggg
     4141 tggggtgggg aaaaggaaga aacgcgggcg tattggcccc aatggggtct cggtggggta
     4201 tcgacagagt gccagccctg ggaccgaacc ccgcgtttat gaacaaacga cccaacaccg
     4261 tgcgttttat tctgtctttt tattgccgtc atagcgcggg ttccttccgg tattgtctcc
     4321 ttccgtgttt cagttagcct ccccctaggg tgggcgaaga actccagcat gagatccccg
     4381 cgctggagga tcatccagcc ggcgtcccgg aaaacgattc cgaagcccaa cctttcatag
     4441 aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg gtcggtcatt
     4501 tcgaacccca gagtcccgct cagaagaact cgtcaagaag gcgatagaag gcgatgcgct
     4561 gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa
     4621 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca
     4681 gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc
     4741 aggcatcgcc atgggtcacg acgagatcct cgccgtcggg catgctcgcc ttgagcctgg
     4801 cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa
     4861 gaccggcttc catccgagta cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg
     4921 ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt
     4981 tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca
     5041 gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg
     5101 tggccagcca cgatagccgc gctgcctcgt cttgcagttc attcagggca ccggacaggt
     5161 cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag
     5221 agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg
     5281 gagaacctgc gtgcaatcca tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt
     5341 gatcgatctt tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc
     5401 tcagaggccg aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatg
//
