LOCUS       sequence-457945-        6122 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein stability reporter construct for transient expression in 
            mammalian cells. Stability of N-terminal GFP-fusion protein can be 
            assessed by flow cytometry by normalizing to mCherry expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6122)
  AUTHORS   Manford AG, Rodriguez-Perez F, Shih KY, Shi Z, Berdan CA, Choe M, 
            Titov DV, Nomura DK, Rape M
  TITLE     A Cellular Mechanism to Detect and Alleviate Reductive Stress.
  JOURNAL   Cell. 2020 Oct 1;183(1):46-61.e21. doi: 10.1016/j.cell.2020.08.034. 
            Epub 2020 Sep 16.
  PUBMED    32941802
REFERENCE   2  (bases 1 to 6122)
  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"; doi: 
            "10.1016/j.cell.2020.08.034"; journalName: "Cell"; date: "2020-10-1-
            1"; volume: "183"; issue: "1"; pages: "46-61.e21"
COMMENT     Sequence Label: pCS2+ GFP-2xESP3I-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..6122
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      85..94
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             91..807
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     misc_feature    853..1425
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1020..1037)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1247..1266
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1434..2144
                     /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(1579..1597)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    2174..2308
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2198..2217)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2252..2271
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2421..2441)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2421..2439)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2460..2476)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2460..2476)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2473..2495)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2484..2500
                     /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(2508..2514,2515..2532,2533..2538))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2553..2574
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2691..2708)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2862..3450)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2942..2961)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3621..4412,4413..4481))
                     /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     4244..4263
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4482..4586)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4612..5067)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4749..4770)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4961..4980
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5138..6122
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcgga tcccgccacc atggtgagca agggcgccga gctgttcacc
      121 ggcatcgtgc ccatcctgat cgagctgaat ggcgatgtga atggccacaa gttcagcgtg
      181 agcggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      241 accggcaagc tgcctgtgcc ctggcccacc ctggtgacca ccctgagcta cggcgtgcag
      301 tgcttctcac gctaccccga tcacatgaag cagcacgact tcttcaagag cgccatgcct
      361 gagggctaca tccaggagcg caccatcttc ttcgaggatg acggcaacta caagtcgcgc
      421 gccgaggtga agttcgaggg cgataccctg gtgaatcgca tcgagctgac cggcaccgat
      481 ttcaaggagg atggcaacat cctgggcaat aagatggagt acaactacaa cgcccacaat
      541 gtgtacatca tgaccgacaa ggccaagaat ggcatcaagg tgaacttcaa gatccgccac
      601 aacatcgagg atggcagcgt gcagctggcc gaccactacc agcagaatac ccccatcggc
      661 gatggccctg tgctgctgcc cgataaccac tacctgtcca cccagagcgc cctgtccaag
      721 gaccccaacg agaagcgcga tcacatgatc tacttcggct tcgtgaccgc cgccgccatc
      781 acccacggca tggatgagct gtacaaggga ggcggaggca gcggagacgg acgaacgtct
      841 cttgatctag agcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga
      901 ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa
      961 tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc
     1021 tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc
     1081 ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg
     1141 cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca
     1201 accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
     1261 cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct
     1321 ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc
     1381 cccgaaccac ggggacgtgg ttttcctttg aaaaacacga taatacctcc ggaatggtga
     1441 gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc aaggtgcaca
     1501 tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     1561 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttcgcct
     1621 gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag caccccgccg
     1681 acatccccga ctacttgaag ctgtccttcc ccgagggctt caagtgggag cgcgtgatga
     1741 acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctgcag gacggcgagt
     1801 tcatctacaa ggtgaagctg cgcggcacca acttcccctc cgacggcccc gtaatgcaga
     1861 agaaaaccat gggctgggag gcctcctccg agcggatgta ccccgaggac ggcgccctga
     1921 agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac gctgaggtca
     1981 agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac gtcaacatca
     2041 agttggacat cacctcccac aacgaggact acaccatcgt ggaacagtac gaacgcgccg
     2101 agggccgcca ctccaccggc ggcatggacg agctgtacaa gtgatctaga actatagtga
     2161 gtcgtattac gtagatccag acatgataag atacattgat gagtttggac aaaccacaac
     2221 tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
     2281 aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca
     2341 ggttcagggg gaggtgtggg aggtttttta attcgcggcc gcggcgccaa tgcattgggc
     2401 ccggtaccca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg
     2461 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     2521 gggagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     2581 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     2641 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     2701 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     2761 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     2821 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     2881 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     2941 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3001 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     3061 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3121 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3181 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3241 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3301 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3361 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3421 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3481 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     3541 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3601 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3661 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3721 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3781 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3841 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3901 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3961 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     4021 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     4081 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     4141 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     4201 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     4261 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     4321 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4381 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4441 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4501 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4561 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa
     4621 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     4681 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     4741 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     4801 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     4861 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     4921 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     4981 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     5041 cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg
     5101 aagggcgatc ggtgcgggcc tcttcgctat tacgccagtc gaccgccaat tcaatatggc
     5161 gtatatggac tcatgccaat tcaatatggt ggatctggac ctgtgccaat tcaatatggc
     5221 gtatatggac tcgtgccaat tcaatatggt ggatctggac cccagccaat tcaatatggc
     5281 ggacttggca ccatgccaat tcaatatggc ggacctggca ctgtgccaac tggggagggg
     5341 tctacttggc acggtgccaa gtttgaggag gggtcttggc cctgtgccaa gtccgccata
     5401 ttgaattggc atggtgccaa taatggcggc catattggct atatgccagg atcaatatat
     5461 aggcaatatc caatatggcc ctatgccaat atggctattg gccaggttca atactatgta
     5521 ttggccctat gccatatagt attccatata tgggttttcc tattgacgta gatagcccct
     5581 cccaatgggc ggtcccatat accatatatg gggcttccta ataccgccca tagccactcc
     5641 cccattgacg tcaatggtct ctatatatgg tctttcctat tgacgtcata tgggcggtcc
     5701 tattgacgta tatggcgcct cccccattga cgtcaattac ggtaaatggc ccgcctggct
     5761 caatgcccat tgacgtcaat aggaccaccc accattgacg tcaatgggat ggctcattgc
     5821 ccattcatat ccgttctcac gccccctatt gacgtcaatg acggtaaatg gcccacttgg
     5881 cagtacatca atatctatta atagtaactt ggcaagtaca ttactattgg aagtacgcca
     5941 gggtacattg gcagtactcc cattgacgtc aatggcggta aatggcccgc gatggctgcc
     6001 aagtacatcc ccattgacgt caatggggag gggcaatgac gcaaatgggc gttccattga
     6061 cgtaaatggg cggtaggcgt gcctaatggg aggtctatat aagcaatgct cgtttaggga
     6121 ac
//
