LOCUS       sequence-139751-        6778 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Mammalian expression of tandem fusion of mClover3-mRuby3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6778)
  AUTHORS   Bajar BT, Wang ES, Lam AJ, Kim BB, Jacobs CL, Howe ES, Davidson MW, 
            Lin MZ, Chu J
  TITLE     Improving brightness and photostability of green and red fluorescent
            proteins for live cell imaging and FRET reporting.
  JOURNAL   Sci Rep. 2016 Feb 16;6:20889. doi: 10.1038/srep20889.
  PUBMED    26879144
REFERENCE   2  (bases 1 to 6778)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2016-02-16"; volume: "6:20889. doi"; pages: " 
            10.1038/srep20889"
COMMENT     Sequence Label: pKanCMV-mClover3-mRuby3
FEATURES             Location/Qualifiers
     source          1..6778
                     /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"
     CDS             866..883
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(926..947)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1534..1555
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1610..2317
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby2, with improved brightness and photostability (Bajar 
                     et al., 2016)"
                     /label=mRuby3
                     /translation="VSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGRPYEGVQTMR
                     IKVIEGGPLPFAFDILATSFMYGSRTFIKYPADIPDFFKQSFPEGFTWERVTRYEDGGV
                     VTVTQDTSLEDGELVYNVKVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTDI
                     ALKVDGGGHLHCNFVTTYRSKKTVGNIKMPGVHAVDHRLERIEESDNETYVVQREVAVA
                     KYSNLGGGMDELYK"
     promoter        complement(2330..2348)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2331..2348)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2368..2385)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2374..2598
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2644..3072
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2731..2750)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2941..2962
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3081..3101)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3086..3416
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3267..3402
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3329..3348
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3483..4277
                     /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(3537..3556)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4147..4166
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4451..4572
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4488..4507)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4542..4561
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4621..4637)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4621..4637)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4634..4656)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4645..4661
                     /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(4669..4675,4676..4693,4694..4699))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4714..4735
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4852..4869)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5023..5611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5103..5122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5782..6573,6574..6642))
                     /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     6405..6424
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6643..6747)
                     /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 gcagagctgg tttagtgaac cgtcagatcc
      841 gctagcgcta ccggtcgcca ccatgcatca tcaccatcac cacgtgagca agggcgagga
      901 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
      961 gttcagcgtc cgcggcgagg gcgagggcga tgccaccaac ggcaagctga ccctgaagtt
     1021 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccttcggcta
     1081 cggcgtggcc tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     1141 cgccatgccc gaaggctacg tccaggagcg caccatctct ttcaaggacg acggtaccta
     1201 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     1261 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaacttcaa
     1321 cagccactac gtctatatca cggccgacaa gcagaagaac tgcatcaagg ctaacttcaa
     1381 gatccgccac aacgttgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     1441 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagcc atcagtccaa
     1501 gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     1561 cgccctggaa agcggcggcg aagatccgat ggtgagcaaa ggcgaagaag tgtctaaggg
     1621 cgaagagctg atcaaggaaa atatgcgtat gaaggtggtc atggaaggtt cggtcaacgg
     1681 ccaccaattc aaatgcacag gtgaaggaga aggcagaccg tacgagggag tgcaaaccat
     1741 gaggatcaaa gtcatcgagg gaggacccct gccatttgcc tttgacattc ttgccacgtc
     1801 gttcatgtat ggcagccgta cctttatcaa gtacccggcc gacatccctg atttctttaa
     1861 acagtccttt cctgagggtt ttacttggga aagagttacg agatacgaag atggtggagt
     1921 cgtcaccgtc acgcaggaca ccagccttga ggatggcgag ctcgtctaca acgtcaaggt
     1981 cagaggggta aactttccct ccaatggtcc cgtgatgcag aagaagacca agggttggga
     2041 gcctaataca gagatgatgt atccagcaga tggtggtctg agaggataca ctgacatcgc
     2101 actgaaagtt gatggtggtg gccatctgca ctgcaacttc gtgacaactt acaggtcaaa
     2161 aaagaccgtc gggaacatca agatgcccgg tgtccatgcc gttgatcacc gcctggaaag
     2221 gatcgaggag agtgacaatg aaacctacgt agtgcaaaga gaagtggcag ttgccaaata
     2281 cagcaacctt ggtggtggca tggacgagct gtacaagtaa gggccctatt ctatagtgtc
     2341 acctaaatgc tagagctcgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     2401 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     2461 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     2521 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     2581 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     2641 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     2701 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     2761 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg catcccttta gggttccgat
     2821 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     2881 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     2941 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     3001 tataagggat tttggggatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     3061 ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc
     3121 cccaggcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     3181 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3241 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     3301 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     3361 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     3421 tcccgggagc ttgtatatcc attttcggat ctgatcaaga gacaggatga ggatcgtttc
     3481 gcatgattga acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat
     3541 tcggctatga ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt
     3601 cagcgcaggg gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac
     3661 tgcaggacga ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg
     3721 tgctcgacgt tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc
     3781 aggatctcct gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa
     3841 tgcggcggct gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc
     3901 gcatcgagcg agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg
     3961 aagagcatca ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg
     4021 acggcgagga tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa
     4081 atggccgctt ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg
     4141 acatagcgtt ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct
     4201 tcctcgtgct ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc
     4261 ttgacgagtt cttctgagcg ggactctggg gttcgaaatg accgaccaag cgacgcccaa
     4321 cctgccatca cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     4381 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     4441 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     4501 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     4561 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     4621 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4681 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4741 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4801 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4861 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4921 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4981 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5041 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5101 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5161 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg
     5221 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5281 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5341 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5401 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5461 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5521 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5581 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5641 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5701 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5761 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5821 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5881 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5941 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6001 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6061 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6121 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6181 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6241 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6301 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6361 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6421 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6481 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6541 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6601 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6661 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6721 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtc
//
