LOCUS       sequence-114654-        6949 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Clover-mRuby2 tandem fusion for FRET calibration.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6949)
  TITLE     Beam Lab unpublished plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6949)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pcDNA3.1-Clover-mRuby2
FEATURES             Location/Qualifiers
     source          1..6949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(947..949,950..952,953..1663)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from GFP (Lam et al., 2012)"
                     /label=Clover
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSALSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(992..1013)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1600..1621
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             join(1697..1705,1706..1720)
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     CDS             1727..2437
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     promoter        complement(2501..2519)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2502..2519)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2539..2556)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2545..2769
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2815..3243
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2902..2921)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3112..3133
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3252..3272)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3257..3587
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3438..3573
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3500..3519
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3654..4448
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3708..3727)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4318..4337
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4622..4743
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4659..4678)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4713..4732
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4792..4808)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4792..4808)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4805..4827)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4816..4832
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4840..4846,4847..4864,4865..4870))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4885..4906
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5023..5040)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5194..5782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5274..5293)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5953..6744,6745..6813))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6576..6595
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6814..6918)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat ccactagtaa cggccgccag tgtgctggaa ttcggcatgg tgagcaaggg
      961 cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg
     1021 ccacaagttc agcgtccgcg gcgagggcga gggcgatgcc accaacggca agctgaccct
     1081 gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccacctt
     1141 cggctacggc gtggcctgct tcagccgcta ccccgaccac atgaagcagc acgacttctt
     1201 caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atctctttca aggacgacgg
     1261 tacctacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga
     1321 gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa
     1381 cttcaacagc cacaacgtct atatcacggc cgacaagcag aagaacggca tcaaggctaa
     1441 cttcaagatc cgccacaacg ttgaggacgg cagcgtgcag ctcgccgacc actaccagca
     1501 gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagccatca
     1561 gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt
     1621 gaccgccgcc gggattacac atggcatgga cgagctgtac aagtctagca tgactggtgg
     1681 acagcaaatg ggtcgggatc tgtacgacga tgacgataag gatccgatgg tgtctaaggg
     1741 cgaagagctg atcaaggaaa atatgcgtat gaaggtggtc atggaaggtt cggtcaacgg
     1801 ccaccaattc aaatgcacag gtgaaggaga aggcaatccg tacatgggaa ctcaaaccat
     1861 gaggatcaaa gtcatcgagg gaggacccct gccatttgcc tttgacattc ttgccacgtc
     1921 gttcatgtat ggcagccgta cttttatcaa gtacccgaaa ggcattcctg atttctttaa
     1981 acagtccttt cctgagggtt ttacttggga aagagttacg agatacgaag atggtggagt
     2041 cgtcaccgtc atgcaggaca ccagccttga ggatggctgt ctcgtttacc acgtccaagt
     2101 cagaggggta aactttccct ccaatggtcc cgtgatgcag aagaagacca agggttggga
     2161 gcctaataca gagatgatgt atccagcaga tggtggtctg aggggataca ctcatatggc
     2221 actgaaagtt gatggtggtg gccatctgtc ttgctctttc gtaacaactt acaggtcaaa
     2281 aaagaccgtc gggaacatca agatgcccgg tatccatgcc gttgatcacc gcctggaaag
     2341 gttagaggaa agtgacaatg aaatgttcgt agtacaacgc gaacacgcag ttgccaagtt
     2401 cgccgggctt ggtggtggga tggacgagct gtacaagtaa gaattctgca gatatccatc
     2461 acactggcgg ccgctcgagc atgcatctag agggccctat tctatagtgt cacctaaatg
     2521 ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc
     2581 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     2641 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     2701 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     2761 gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc
     2821 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     2881 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     2941 ccggctttcc ccgtcaagct ctaaatcggg gcatcccttt agggttccga tttagtgctt
     3001 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     3061 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     3121 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     3181 ttttggggat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     3241 attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccaggcag
     3301 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     3361 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     3421 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     3481 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3541 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3601 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
     3661 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3721 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3781 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     3841 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3901 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3961 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     4021 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     4081 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     4141 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     4201 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     4261 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     4321 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     4381 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     4441 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     4501 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     4561 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     4621 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4681 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4741 ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     4801 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4861 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4921 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4981 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     5041 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     5101 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5161 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5221 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5281 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5341 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg
     5401 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5461 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5521 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5581 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt
     5641 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5701 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5761 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5821 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5881 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5941 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     6001 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     6061 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6121 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6181 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6241 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6301 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6361 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6421 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6481 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6541 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6601 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6661 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6721 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6781 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6841 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6901 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
