LOCUS       sequence-220241-        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)
  TITLE     Gadella lab plasmids
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"
COMMENT     Sequence Label: mCherry-T2A-LSS-SGFP2
FEATURES             Location/Qualifiers
     source          1..5456
                     /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             613..1320
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(758..776)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1026..1045
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1327..1380
                     /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      1396..1405
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1402..1404,1405..1407,1408..2118)
                     /codon_start=1
                     /product="strongly enhanced green fluorescent protein 
                     (Kremers et al., 2007)"
                     /label=SGFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSIQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1447..1468)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1708..1727)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2055..2076
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2244..2365
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2281..2300)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2335..2354
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2372..2827)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2509..2530)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2721..2740
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2854..2958
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2960..3317
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2960..3151
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2983..3003)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3168..3303
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3352..4146
                     /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             3352..4146
                     /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(3406..3425)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4016..4035
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4334..4353)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4378..4425
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4754..5342
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5243..5262
                     /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 gaggataaca tggccatcat caaggagttc
      661 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
      721 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
      781 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
      841 tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc cgagggcttc
      901 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
      961 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     1021 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     1081 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     1141 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     1201 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     1261 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     1321 tccggagagg gcagaggaag tcttctaaca tgcggtgacg tggaggagaa tcccggccct
     1381 ggggatccac cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg
     1441 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
     1501 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
     1561 ctgcccgtgc cctggcccac cctcgtgacc accctgagct acggcgtgca gtgcttcgcc
     1621 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
     1681 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     1741 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     1801 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     1861 accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacatcgag
     1921 gacggcggcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1981 gtgctgctgc ccgacaacca ctacctgagc atccagtcca agctgagcaa agaccccaac
     2041 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     2101 atggacgagc tgtacaagta aagcggccgc gactctagat cataatcagc cataccacat
     2161 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     2221 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     2281 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     2341 tgtccaaact catcaatgta tcttaaggcg taaattgtaa gcgttaatat tttgttaaaa
     2401 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     2461 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     2521 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     2581 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt
     2641 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     2701 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     2761 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     2821 ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     2881 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     2941 tattgaaaaa ggaagagtcc tgaggcggaa agaaccagct gtggaatgtg tgtcagttag
     3001 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3061 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     3121 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     3181 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     3241 aggccgaggc cgcctcggcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     3301 gcctaggctt ttgcaaagat cgatcaagag acaggatgag gatcgtttcg catgattgaa
     3361 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     3421 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     3481 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaagacgag
     3541 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     3601 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     3661 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     3721 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     3781 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     3841 gggctcgcgc cagccgaact gttcgccagg ctcaaggcga gcatgcccga cggcgaggat
     3901 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     3961 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4021 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4081 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4141 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4201 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4261 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccta
     4321 gggggaggct aactgaaaca cggaaggaga caataccgga aggaacccgc gctatgacgg
     4381 caataaaaag acagaataaa acgcacggtg ttgggtcgtt tgttcataaa cgcggggttc
     4441 ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc caatacgccc
     4501 gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca gggctcgcag
     4561 ccaacgtcgg ggcggcaggc cctgccatag cctcaggtta ctcatatata ctttagattg
     4621 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4681 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4741 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4801 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4861 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4921 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4981 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5041 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5101 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5161 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5221 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5281 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5341 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5401 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc atgcat
//
