LOCUS       sequence-153227-        6878 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression N-term FLAG and C-term mCherry hPPP1R15B (CReP 
            4-713).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6878)
  AUTHORS   Chen R, Rato C, Yan Y, Crespillo-Casado A, Clarke HJ, Harding HP, 
            Marciniak SJ, Read RJ, Ron D
  TITLE     G-actin provides substrate-specificity to eukaryotic initiation 
            factor 2alpha holophosphatases.
  JOURNAL   Elife. 2015 Mar 16;4. doi: 10.7554/eLife.04871.
  PUBMED    25774600
REFERENCE   2  (bases 1 to 6878)
  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: "Elife."; 
            date: "2015-03-16"; volume: "4. doi"; pages: " 10.7554/eLife.04871"
COMMENT     Sequence Label: FLAG_hPPP1R15B_4-713_mCherry_UK1298
FEATURES             Location/Qualifiers
     source          1..6878
                     /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"
     CDS             654..677
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      2829..2838
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2835..3545
                     /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(2980..2998)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3248..3267
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     polyA_signal    3666..3787
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3703..3722)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3757..3776
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3794..4249)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3931..3952)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4143..4162
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4276..4380
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4382..4739
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4382..4573
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4405..4425)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4590..4725
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4774..5568
                     /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             4774..5568
                     /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(4828..4847)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5438..5457
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5756..5775)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5800..5847
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      6176..6764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6665..6684
                     /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 ccggactcag atctcgagct cgtttagtga accgtcagaa ttgatctacc atggactaca
      661 aagacgatga cgacaagctt gggacaggcg gatcgcggaa acggcttggc cctcgggcgg
      721 gcttccggtt ctggccaccc tttttccctc ggcgatcgca agcaggctct tctaagttcc
      781 cgacgcctct tggcccggaa aactccggga accccacact gctttcctct gcccagcccg
      841 agactcgggt cagttactgg acgaaactgc tctcccagct ccttgcgccg ctccccggat
      901 tgcttcagaa ggtgctaatt tggagccaac ttttcggtgg aatgtttccg accagatggc
      961 tagattttgc tggagtctac agcgccctga gagccctgaa gggacgggag aaaccagccg
     1021 cccccacagc gcagaaatct ttgagttcgc tgcagctcga ctcctcagac ccctcggtca
     1081 ccagtcccct tgattggcta gaggagggga tccactggca atactcgccc ccagacctaa
     1141 aattggagct taaggccaag ggaagtgctt tggaccctgc agcacaggct tttctcttag
     1201 agcagcagct gtggggagtg gagctgttgc ccagtagcct tcaatcccgt ctgtactcta
     1261 accgggaact tggctcttcg ccctctgggc ctctaaacat tcaacgcata gacaatttca
     1321 gtgtggtatc ctatttgctg aacccttcct acctggactg ctttcctagg ctagaagtca
     1381 gctatcagaa cagtgatgga aatagcgagg tagtcggctt ccagacacta accccagaga
     1441 gcagctgcct gagagaggac cattgtcatc cccagccgct gagtgcagaa ctcattccgg
     1501 cctcgtggca gggatgtcca cctctttcta cggaaggcct accagaaatt caccatcttc
     1561 gcatgaaacg gctggaattc cttcaacagg ctagcaaggg gcaagattta cccacccctg
     1621 accaggataa tggctaccac agcctggagg aggaacacag ccttctccgg atggatccaa
     1681 aacactgcag agataaccca acacagtttg ttcctgctgc tggagacatt cctggaaaca
     1741 cccaggaatc cactgaagaa aaaatagaat tattaactac agaggttcca cttgctttgg
     1801 aagaagagag cccttctgag ggctgtccat ctagtgagat acctatggaa aaggagcctg
     1861 gagagggccg aataagtgta gttgattact catacctaga aggtgacctt cccatttctg
     1921 ccagaccagc ttgtagtaac aaactgatag attatatttt gggaggtgca tccagtgacc
     1981 tggaaacaag ttctgatcca gaaggtgagg attgggatga ggaagctgag gatgatggtt
     2041 ttgatagtga tagctcactg tcagactcag accttgaaca agaccctgaa gggcttcacc
     2101 tttggaactc tttctgcagt gtagatcctt ataatcccca gaactttaca gcaacaattc
     2161 agactgctgc cagaattgtt cctgaagagc cttctgattc agagaaggat ttgtctggca
     2221 agtctgatct agagaattcc tcccagtctg gaagccttcc tgagacccct gagcatagtt
     2281 ctggggagga agatgactgg gaatctagtg cagatgaagc agagagtctc aaactgtgga
     2341 actcattctg taattctgat gacccctaca accctttaaa ttttaaggct ccttttcaaa
     2401 catcagggga aaatgagaaa ggctgtcgtg actcaaagac cccatctgag tccattgtgg
     2461 ccatttctga gtgtcacacc ttactttctt gtaaggtgca gctgttgggg agccaagaaa
     2521 gtgaatgtcc agactcggta cagcgtgacg ttctttctgg aggaagacac acacatgtca
     2581 aaagaaaaaa ggtaaccttc cttgaagaag ttactgagta ttatataagt ggtgatgagg
     2641 atcgcaaagg accatgggaa gaatttgcaa gggatggatg caggttccag aaacgaattc
     2701 aagaaacaga agatgctatt ggatattgct tgacatttga acacagagaa agaatgttta
     2761 atagactcca gggaacatgc ttcaaaggac ttaatgttct caagcaatgt gtcgacggta
     2821 gatccatcgc caccatggtg agcaagggcg aggaggataa catggccatc atcaaggagt
     2881 tcatgcgctt caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg
     2941 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     3001 gtggccccct gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg
     3061 cctacgtgaa gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct
     3121 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact
     3181 cctccctgca ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct
     3241 ccgacggccc cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt
     3301 accccgagga cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg
     3361 gccactacga cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg
     3421 gcgcctacaa cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg
     3481 tggaacagta cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     3541 agtagcggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg
     3601 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     3661 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3721 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3781 tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     3841 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     3901 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     3961 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     4021 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     4081 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     4141 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     4201 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     4261 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4321 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4381 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     4441 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     4501 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     4561 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4621 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4681 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     4741 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     4801 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     4861 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     4921 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     4981 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     5041 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     5101 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     5161 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     5221 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     5281 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     5341 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     5401 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     5461 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     5521 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     5581 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     5641 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     5701 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     5761 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     5821 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     5881 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     5941 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     6001 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt
     6061 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6121 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6181 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6241 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6301 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     6361 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6421 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6481 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6541 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6601 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6661 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6721 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6781 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6841 cccctgattc tgtggataac cgtattaccg ccatgcat
//
