LOCUS       sequence-458881-        6177 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein stability reporter construct for CDK5R1 consisting of aa 
            283-307 for transient overexpression in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6177)
  AUTHORS   Manford AG, Mena EL, Shih KY, Gee CL, McMinimy R, Martinez-Gonzalez 
            B, Sherriff R, Lew B, Zoltek M, Rodriguez-Perez F, Woldesenbet M, 
            Kuriyan J, Rape M
  TITLE     Structural basis and regulation of the reductive stress response.
  JOURNAL   Cell. 2021 Oct 14;184(21):5375-5390.e16. doi: 
            10.1016/j.cell.2021.09.002. Epub 2021 Sep 24.
  PUBMED    34562363
REFERENCE   2  (bases 1 to 6177)
  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.2021.09.002"; journalName: "Cell"; date: 
            "2021-10-14- 14"; volume: "184"; issue: "21"; pages: "5375-5390.e16"
COMMENT     Sequence Label: pCS2+ GFP-CDK5R1 degron (283-307)-IRES-mCherry
FEATURES             Location/Qualifiers
     source          1..6177
                     /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    908..1480
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1075..1092)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1302..1321
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1489..2199
                     /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(1634..1652)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    2229..2363
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2253..2272)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2307..2326
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2476..2496)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2476..2494)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2515..2531)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2515..2531)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2528..2550)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2539..2555
                     /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(2563..2569,2570..2587,2588..2593))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2608..2629
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2746..2763)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2917..3505)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2997..3016)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3676..4467,4468..4536))
                     /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     4299..4318
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4537..4641)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4667..5122)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4804..4825)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5016..5035
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5193..6177
                     /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 gcacacaggt cttctccgac
      841 ctgaagaacg agagcggcca ggaggacaag aagcggctcc tcctaggcct ggatcggtga
      901 tctagagccc ctctccctcc ccccccccta acgttactgg ccgaagccgc ttggaataag
      961 gccggtgtgc gtttgtctat atgttatttt ccaccatatt gccgtctttt ggcaatgtga
     1021 gggcccggaa acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg
     1081 ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt
     1141 gaagacaaac aacgtctgta gcgacccttt gcaggcagcg gaacccccca cctggcgaca
     1201 ggtgcctctg cggccaaaag ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc
     1261 agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa atggctctcc tcaagcgtat
     1321 tcaacaaggg gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc
     1381 ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta ggccccccga
     1441 accacgggga cgtggttttc ctttgaaaaa cacgataata cctccggaat ggtgagcaag
     1501 ggcgaggagg ataacatggc catcatcaag gagttcatgc gcttcaaggt gcacatggag
     1561 ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg cccctacgag
     1621 ggcacccaga ccgccaagct gaaggtgacc aagggtggcc ccctgccctt cgcctgggac
     1681 atcctgtccc ctcagttcat gtacggctcc aaggcctacg tgaagcaccc cgccgacatc
     1741 cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt gatgaacttc
     1801 gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg cgagttcatc
     1861 tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat gcagaagaaa
     1921 accatgggct gggaggcctc ctccgagcgg atgtaccccg aggacggcgc cctgaagggc
     1981 gagatcaagc agaggctgaa gctgaaggac ggcggccact acgacgctga ggtcaagacc
     2041 acctacaagg ccaagaagcc cgtgcagctg cccggcgcct acaacgtcaa catcaagttg
     2101 gacatcacct cccacaacga ggactacacc atcgtggaac agtacgaacg cgccgagggc
     2161 cgccactcca ccggcggcat ggacgagctg tacaagtgat ctagaactat agtgagtcgt
     2221 attacgtaga tccagacatg ataagataca ttgatgagtt tggacaaacc acaactagaa
     2281 tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta tttgtaacca
     2341 ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg tttcaggttc
     2401 agggggaggt gtgggaggtt ttttaattcg cggccgcggc gccaatgcat tgggcccggt
     2461 acccagcttt tgttcccttt agtgagggtt aattgcgcgc ttggcgtaat catggtcata
     2521 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccgggag
     2581 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     2641 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     2701 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     2761 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2821 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2881 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2941 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3001 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3061 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3121 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3181 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3241 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3301 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3361 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3421 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3481 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3541 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3601 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3661 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3721 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3781 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3841 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3901 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3961 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4021 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4081 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4141 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4201 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4261 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4321 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4381 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4441 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4501 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4561 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4621 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt
     4681 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     4741 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     4801 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     4861 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     4921 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     4981 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     5041 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     5101 aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg ttgggaaggg
     5161 cgatcggtgc gggcctcttc gctattacgc cagtcgaccg ccaattcaat atggcgtata
     5221 tggactcatg ccaattcaat atggtggatc tggacctgtg ccaattcaat atggcgtata
     5281 tggactcgtg ccaattcaat atggtggatc tggaccccag ccaattcaat atggcggact
     5341 tggcaccatg ccaattcaat atggcggacc tggcactgtg ccaactgggg aggggtctac
     5401 ttggcacggt gccaagtttg aggaggggtc ttggccctgt gccaagtccg ccatattgaa
     5461 ttggcatggt gccaataatg gcggccatat tggctatatg ccaggatcaa tatataggca
     5521 atatccaata tggccctatg ccaatatggc tattggccag gttcaatact atgtattggc
     5581 cctatgccat atagtattcc atatatgggt tttcctattg acgtagatag cccctcccaa
     5641 tgggcggtcc catataccat atatggggct tcctaatacc gcccatagcc actcccccat
     5701 tgacgtcaat ggtctctata tatggtcttt cctattgacg tcatatgggc ggtcctattg
     5761 acgtatatgg cgcctccccc attgacgtca attacggtaa atggcccgcc tggctcaatg
     5821 cccattgacg tcaataggac cacccaccat tgacgtcaat gggatggctc attgcccatt
     5881 catatccgtt ctcacgcccc ctattgacgt caatgacggt aaatggccca cttggcagta
     5941 catcaatatc tattaatagt aacttggcaa gtacattact attggaagta cgccagggta
     6001 cattggcagt actcccattg acgtcaatgg cggtaaatgg cccgcgatgg ctgccaagta
     6061 catccccatt gacgtcaatg gggaggggca atgacgcaaa tgggcgttcc attgacgtaa
     6121 atgggcggta ggcgtgccta atgggaggtc tatataagca atgctcgttt agggaac
//
