LOCUS       sequence-458623-        7487 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MCRS1 stability reporter construct for transient expression in 
            mammalian cells. Stability of GFP-fusion protein can be assed by 
            flow cytometry by normalizing to mCherry expression..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7487)
  AUTHORS   Mark KG, Kolla S, Aguirre JD, Garshott DM, Schmitt S, Haakonsen DL, 
            Xu C, Kater L, Kempf G, Martinez-Gonzalez B, Akopian D, See SK, 
            Thoma NH, Rape M
  TITLE     Orphan quality control shapes network dynamics and gene expression.
  JOURNAL   Cell. 2023 Aug 3;186(16):3460-3475.e23. doi: 
            10.1016/j.cell.2023.06.015. Epub 2023 Jul 20.
  PUBMED    37478862
REFERENCE   2  (bases 1 to 7487)
  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.2023.06.015"; journalName: "Cell"; date: "2023-08-3-
            3"; volume: "186"; issue: "16"; pages: "3460-3475.e23"
COMMENT     Sequence Label: pCS2-MCRS1-GFP-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..7487
                     /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             1492..2211
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     misc_feature    2219..2791
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2386..2403)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2613..2632
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     primer_bind     complement(2945..2963)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    3539..3673
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3563..3582)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3617..3636
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3786..3806)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3786..3804)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3825..3841)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3825..3841)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3838..3860)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3849..3865
                     /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(3873..3879,3880..3897,3898..3903))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3918..3939
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4056..4073)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4227..4815)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4307..4326)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4986..5777,5778..5846))
                     /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     5609..5628
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5847..5951)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5977..6432)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6114..6135)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6326..6345
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        6503..7487
                     /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 atggacaaag attctcaggg gctgctagat
      121 tcatccctga tggcatcagg cactgccagc cgctcagagg atgaggagtc actggcaggg
      181 cagaagcgag cctcctccca ggccttgggc accatcccta aacggagaag ctcctccagg
      241 ttcatcaaga ggaagaagtt cgatgatgag ctggtggaga gcagcctggc aaaatcttct
      301 acccgggcaa agggggccag tggggtggaa ccagggcgct gttcggggag tgaaccctcc
      361 tccagtgaga agaagaaggt atccaaagcc cccagcactc ctgtgccacc cagcccagcc
      421 ccagcccctg gactcaccaa gcgtgtgaag aagagtaaac agccacttca ggtgaccaag
      481 gatctgggcc gctggaagcc tgcagatgac ctcctgctca taaatgctgt gttgcagacc
      541 aacgacctga cctccgtcca cctgggcgtg aaattcagct gccgcttcac ccttcgggag
      601 gtccaggagc gttggtacgc cctgctctac gatcctgtca tctccaagtt ggcctgtcag
      661 gccatgaggc agctgcaccc agaggctatt gcagccatcc agagcaaggc cctgtttagc
      721 aaggctgagg agcagctgct gagcaaagtg ggatcgacca gccagcccac cttggagacc
      781 ttccaggacc tgctgcacag acaccctgat gccttctacc tggcccgtac cgcgaaggcc
      841 ctgcaggccc actggcagct catgaagcag tattacctgc tggaggacca gacagtgcag
      901 ccgctgccca aaggggacca agtgctgaac ttctctgatg cagaggacct gattgatgac
      961 agtaagctca aggacatgcg agatgaggtc ctggaacatg agctgatggt ggctgaccgg
     1021 cgccagaagc gagagattcg gcagctggaa caggaactgc ataagtggca ggtgctagtg
     1081 gacagcatca caggcatgag ctctccggac ttcgacaacc agacactggc agtgctgcgg
     1141 ggccgcatgg tgcggtacct gatgcgctcg cgtgagatca ccctgggcag agcaaccaag
     1201 gataaccaga ttgatgtgga cctgtctctg gagggtccgg cctggaagat atcccggaaa
     1261 caaggtgtca tcaagctgaa gaacaacggt gatttcttca ttgccaatga gggtcgacgg
     1321 cccatctaca tcgatggacg gccggtgctc tgtggctcca aatggcgcct cagcaacaac
     1381 tctgtggtgg agatcgccag cctgcgattc gtcttcctta tcaaccagga cctcattgcc
     1441 ctcatcaggg ctgaggctgc caagatcaca ccacagggag gcggaggcag catggtgagc
     1501 aagggcgccg agctgttcac cggcatcgtg cccatcctga tcgagctgaa tggcgatgtg
     1561 aatggccaca agttcagcgt gagcggcgag ggcgagggcg atgccaccta cggcaagctg
     1621 accctgaagt tcatctgcac caccggcaag ctgcctgtgc cctggcccac cctggtgacc
     1681 accctgagct acggcgtgca gtgcttctca cgctaccccg atcacatgaa gcagcacgac
     1741 ttcttcaaga gcgccatgcc tgagggctac atccaggagc gcaccatctt cttcgaggat
     1801 gacggcaact acaagtcgcg cgccgaggtg aagttcgagg gcgataccct ggtgaatcgc
     1861 atcgagctga ccggcaccga tttcaaggag gatggcaaca tcctgggcaa taagatggag
     1921 tacaactaca acgcccacaa tgtgtacatc atgaccgaca aggccaagaa tggcatcaag
     1981 gtgaacttca agatccgcca caacatcgag gatggcagcg tgcagctggc cgaccactac
     2041 cagcagaata cccccatcgg cgatggccct gtgctgctgc ccgataacca ctacctgtcc
     2101 acccagagcg ccctgtccaa ggaccccaac gagaagcgcg atcacatgat ctacttcggc
     2161 ttcgtgaccg ccgccgccat cacccacggc atggatgagc tgtacaagtg atctagagcc
     2221 cctctccctc ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg
     2281 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga
     2341 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa
     2401 tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa
     2461 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct
     2521 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg
     2581 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg
     2641 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca
     2701 catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg
     2761 acgtggtttt cctttgaaaa acacgataat acctccggaa tggtgagcaa gggcgaggag
     2821 gataacatgg ccatcatcaa ggagttcatg cgcttcaagg tgcacatgga gggctccgtg
     2881 aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag
     2941 accgccaagc tgaaggtgac caagggtggc cccctgccct tcgcctggga catcctgtcc
     3001 cctcagttca tgtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac
     3061 ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc
     3121 ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg
     3181 aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa aaccatgggc
     3241 tgggaggcct cctccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag
     3301 cagaggctga agctgaagga cggcggccac tacgacgctg aggtcaagac cacctacaag
     3361 gccaagaagc ccgtgcagct gcccggcgcc tacaacgtca acatcaagtt ggacatccct
     3421 cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc cgccactcca
     3481 ccggcggcat ggacgagctg tacaagtgat ctagaactat agtgagtcgt attacgtaga
     3541 tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa
     3601 aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg
     3661 caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt
     3721 gtgggaggtt ttttaattcg cggccgcggc gccaatgcat tgggcccggt acccagcttt
     3781 tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata gctgtttcct
     3841 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccgggag cataaagtgt
     3901 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     3961 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4021 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4081 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4141 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4201 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4261 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4321 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4381 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4441 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4501 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4561 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4621 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4681 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4741 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4801 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4861 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4921 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4981 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5041 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5101 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5161 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5221 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5281 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5341 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5401 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5461 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5521 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5581 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5641 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5701 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5761 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5821 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5881 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5941 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     6001 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     6061 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     6121 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     6181 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     6241 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     6301 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     6361 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     6421 tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
     6481 gggcctcttc gctattacgc cagtcgaccg ccaattcaat atggcgtata tggactcatg
     6541 ccaattcaat atggtggatc tggacctgtg ccaattcaat atggcgtata tggactcgtg
     6601 ccaattcaat atggtggatc tggaccccag ccaattcaat atggcggact tggcaccatg
     6661 ccaattcaat atggcggacc tggcactgtg ccaactgggg aggggtctac ttggcacggt
     6721 gccaagtttg aggaggggtc ttggccctgt gccaagtccg ccatattgaa ttggcatggt
     6781 gccaataatg gcggccatat tggctatatg ccaggatcaa tatataggca atatccaata
     6841 tggccctatg ccaatatggc tattggccag gttcaatact atgtattggc cctatgccat
     6901 atagtattcc atatatgggt tttcctattg acgtagatag cccctcccaa tgggcggtcc
     6961 catataccat atatggggct tcctaatacc gcccatagcc actcccccat tgacgtcaat
     7021 ggtctctata tatggtcttt cctattgacg tcatatgggc ggtcctattg acgtatatgg
     7081 cgcctccccc attgacgtca attacggtaa atggcccgcc tggctcaatg cccattgacg
     7141 tcaataggac cacccaccat tgacgtcaat gggatggctc attgcccatt catatccgtt
     7201 ctcacgcccc ctattgacgt caatgacggt aaatggccca cttggcagta catcaatatc
     7261 tattaatagt aacttggcaa gtacattact attggaagta cgccagggta cattggcagt
     7321 actcccattg acgtcaatgg cggtaaatgg cccgcgatgg ctgccaagta catccccatt
     7381 gacgtcaatg gggaggggca atgacgcaaa tgggcgttcc attgacgtaa atgggcggta
     7441 ggcgtgccta atgggaggtc tatataagca atgctcgttt agggaac
//
