LOCUS       sequence-403936-        6793 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-His-tagged CDK5RAP3 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6793)
  AUTHORS   Ishimura R, El-Gowily AH, Noshiro D, Komatsu-Hirota S, Ono Y, Shindo
            M, Hatta T, Abe M, Uemura T, Lee-Okada HC, Mohamed TM, Yokomizo T, 
            Ueno T, Sakimura K, Natsume T, Sorimachi H, Inada T, Waguri S, Noda 
            NN, Komatsu M
  TITLE     The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.
  JOURNAL   Nat Commun. 2022 Dec 21;13(1):7857. doi: 10.1038/s41467-022-35501-0.
  PUBMED    36543799
REFERENCE   2  (bases 1 to 6793)
  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: "Nat 
            Commun."; date: "2022-12-21"; volume: "13(1):7857. doi"; pages: " 
            10.1038/s41467-022-35501-0"
COMMENT     Sequence Label: pIRESpuroN3xFLAG-His-CDK5RAP3
FEATURES             Location/Qualifiers
     source          1..6793
                     /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"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             979..1044
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1051..1068
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     intron          2680..2909
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2892..2911
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    join(2967..3541,3542..3544,3545..3553)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3134..3151)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3361..3380
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3573..4172
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3573..3592)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4069..4089
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4410..4531
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4447..4466)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4636..4652)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4636..4652)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4649..4671)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4660..4676
                     /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(4684..4690,4691..4708,4709..4714))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4729..4750
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4867..4884)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5038..5626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5118..5137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5797..6588,6589..6657))
                     /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     6420..6439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6658..6762)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct 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 atgggtggag 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 gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcggat cgatatctgc ggcctagcta gcgcttaagg cctgttaacc ggtcgtacgt
      961 ctccggattc gaattatgga ctacaaagac catgacggtg attataaaga tcatgacatc
     1021 gattacaagg atgacgatga caaggaattg catcatcacc atcaccacga attcggatcc
     1081 tctagaatgg aggaccatca gcacgtgccc atcgacatcc agaccagcaa gctgctcgat
     1141 tggctggtgg acagaaggca ctgcagcctg aaatggcaga gtctggtgct gacgatccgc
     1201 gagaagatca atgctgccat ccaggacatg ccagagagcg aagagatcgc ccagctgctg
     1261 tctgggtcct acattcacta ctttcactgc ctaagaatcc tggaccttct caaaggcaca
     1321 gaggcctcca cgaagaatat ttttggccga tactcttcac agcggatgaa ggattggcag
     1381 gagattatag ctctgtatga gaaggacaac acctacttag tggaactctc tagcctcctg
     1441 gttcggaatg tcaactatga gatcccctca ctgaagaagc agattgccaa gtgccagcag
     1501 ctgcagcaag aatacagccg caaggaggag gagtgccagg caggggctgc cgagatgcgg
     1561 gagcagttct accactcctg caagcagtat ggcatcacgg gcgaaaatgt ccgaggagaa
     1621 ctgctggccc tggtgaagga cctgccgagt cagctggctg agattggggc agcggctcag
     1681 cagtccctgg gggaagccat tgacgtgtac caggcgtctg tggggtttgt gtgtgagagc
     1741 cccacagagc aggtgttgcc aatgctgcgg ttcgtgcaga agcggggaaa ctcaacggtg
     1801 tacgagtgga ggacagggac agagccctct gtggtggaac gaccccacct cgaggagctt
     1861 cctgagcagg tggcagaaga tgcgattgac tggggcgact ttggggtaga ggcagtgtct
     1921 gaggggactg actctggcat ctctgccgag gctgctggaa tcgactgggg catcttcccg
     1981 gaatcagatt caaaggatcc tggaggtgat gggatagact ggggagacga tgctgttgct
     2041 ttgcagatca cagtgctgga agcaggaacc caggctccag aaggtgttgc caggggccca
     2101 gatgccctga cactgcttga atacactgag acccggaatc agttccttga tgagctcatg
     2161 gagcttgaga tcttcttagc ccagagagca gtggagttga gtgaggaggc agatgtcctg
     2221 tctgtgagcc agttccagct ggctccagcc atcctgcagg gccagaccaa agagaagatg
     2281 gttaccatgg tgtcagtgct ggaggatctg attggcaagc ttaccagtct tcagctgcaa
     2341 cacctgttta tgatcctggc ctcaccaagg tatgtggacc gagtgactga attcctccag
     2401 caaaagctga agcagtccca gctgctggct ttgaagaaag agctgatggt gcagaagcag
     2461 caggaggcac ttgaggagca ggcggctctg gagcctaagc tggacctgct actggagaag
     2521 accaaggagc tgcagaagct gattgaagct gacatctcca agaggtacag cgggcgccct
     2581 gtgaacctga tgggaacctc tctgtgagtc gagggccgca tagataactg atccagtgtg
     2641 ctggaattaa ttcgctgtct gcgagggcca gctgttgggg tgagtactcc ctctcaaaag
     2701 cgggcatgac ttctgcgcta agattgtcag tttccaaaaa cgaggaggat ttgatattca
     2761 cctggcccgc ggtgatgcct ttgagggtgg ccgcgtccat ctggtcagaa aagacaatct
     2821 ttttgttgtc aagcttgagg tgtggcaggc ttgagatctg gccatacact tgagtgacaa
     2881 tgacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac tgcaggtcga
     2941 gcatgcatct agggcggcca attccgcccc tctccctccc ccccccctaa cgttactggc
     3001 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg
     3061 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
     3121 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
     3181 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
     3241 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
     3301 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
     3361 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
     3421 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa
     3481 aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa
     3541 gcttgccaca acccacaagg agacgacctt ccatgaccga gtacaagccc acggtgcgcc
     3601 tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact
     3661 accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc
     3721 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     3781 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     3841 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     3901 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     3961 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     4021 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     4081 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     4141 cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga
     4201 ccgaaaggag cgcacgaccc catggctccg accgaagcca cccggggcgg ccccgccgac
     4261 cccgcacccg cccccgaggc ccaccgactc tagctagata actgatcata atcagccata
     4321 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     4381 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     4441 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4501 gtggtttgtc caaactcatc aatgtatctt aacgctcgat cgagtgcatt ctagttgtgg
     4561 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4621 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4681 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4741 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4801 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4861 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4921 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4981 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5041 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5101 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5161 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5221 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5281 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5341 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5401 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5461 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5521 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5581 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5641 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     5701 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     5761 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5821 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5881 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5941 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6001 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6061 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6121 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6181 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6241 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6301 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6361 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6421 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6481 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6541 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6601 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6661 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     6721 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6781 gccacctgac gtc
//
