LOCUS       sequence-403940-        6237 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses hAG-tagged CDK5RAP3 W269A W294A W312A in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6237)
  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 6237)
  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: phAG-MCL-CDK5RAP3 W269A W294A W312A
FEATURES             Location/Qualifiers
     source          1..6237
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..678
                     /codon_start=1
                     /product="humanized monomeric Azami-Green fluorescent 
                     protein"
                     /label=hmAzami-Green
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNV
                     TEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTA
                     TSNISMRGDCFFYDIRFDGVNFPPNGPVMQKKTLKWEPSTEKMYVRDGVLKGDVNMALL
                     LEGGGHYRCDFKTTYKAKKDVRLPDYHFVDHRIEILKHDKDYNKVKLYENAVARYSMLP
                     SQAK"
     polyA_signal    2421..2542
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2458..2477)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2512..2531
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2549..3004)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2686..2707)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2898..2917
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3031..3135
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3137..3494
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3137..3328
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3160..3180)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3345..3480
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3528..4322
                     /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             3528..4322
                     /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(3582..3601)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4192..4211
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4510..4529)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4554..4601
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4930..5518
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5419..5438
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        5693..5996
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5997..6200
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6151..6171
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      join(6232..6237,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggtgagcg tgatcaagcc cgagatgaag atcaagctgt gcatgagggg caccgtgaac
       61 ggccacaact tcgtgatcga gggcgagggc aagggcaacc cctacgaggg cacccagatc
      121 ctggacctga acgtgaccga gggcgccccc ctgcccttcg cctacgacat cctgaccacc
      181 gtgttccagt acggcaacag ggccttcacc aagtaccccg ccgacatcca ggactacttc
      241 aagcagacct tccccgaggg ctaccactgg gagaggagca tgacctacga ggaccagggc
      301 atctgcaccg ccaccagcaa catcagcatg aggggcgact gcttcttcta cgacatcagg
      361 ttcgacggcg tgaacttccc ccccaacggc cccgtgatgc agaagaagac cctgaagtgg
      421 gagcccagca ccgagaagat gtacgtgagg gacggcgtgc tgaagggcga cgtgaacatg
      481 gccctgctgc tggagggcgg cggccactac aggtgcgact tcaagaccac ctacaaggcc
      541 aagaaggacg tgaggctgcc cgactaccac ttcgtggacc acaggatcga gatcctgaag
      601 cacgacaagg actacaacaa ggtgaagctg tacgagaacg ccgtggccag gtacagcatg
      661 ctgcccagcc aggccaagac cggtaattcc gctgacggcg gcggaggatc gggtggtagt
      721 ggtggttcag gaggaggatc gacccaagga ggatccatgg aggaccatca gcacgtgccc
      781 atcgacatcc agaccagcaa gctgctcgat tggctggtgg acagaaggca ctgcagcctg
      841 aaatggcaga gtctggtgct gacgatccgc gagaagatca atgctgccat ccaggacatg
      901 ccagagagcg aagagatcgc ccagctgctg tctgggtcct acattcacta ctttcactgc
      961 ctaagaatcc tggaccttct caaaggcaca gaggcctcca cgaagaatat ttttggccga
     1021 tactcttcac agcggatgaa ggattggcag gagattatag ctctgtatga gaaggacaac
     1081 acctacttag tggaactctc tagcctcctg gttcggaatg tcaactatga gatcccctca
     1141 ctgaagaagc agattgccaa gtgccagcag ctgcagcaag aatacagccg caaggaggag
     1201 gagtgccagg caggggctgc cgagatgcgg gagcagttct accactcctg caagcagtat
     1261 ggcatcacgg gcgaaaatgt ccgaggagaa ctgctggccc tggtgaagga cctgccgagt
     1321 cagctggctg agattggggc agcggctcag cagtccctgg gggaagccat tgacgtgtac
     1381 caggcgtctg tggggtttgt gtgtgagagc cccacagagc aggtgttgcc aatgctgcgg
     1441 ttcgtgcaga agcggggaaa ctcaacggtg tacgagtgga ggacagggac agagccctct
     1501 gtggtggaac gaccccacct cgaggagctt cctgagcagg tggcagaaga tgcgattgac
     1561 gcgggcgact ttggggtaga ggcagtgtct gaggggactg actctggcat ctctgccgag
     1621 gctgctggaa tcgacgcggg catcttcccg gaatcagatt caaaggatcc tggaggtgat
     1681 gggatagacg cgggagacga tgctgttgct ttgcagatca cagtgctgga agcaggaacc
     1741 caggctccag aaggtgttgc caggggccca gatgccctga cactgcttga atacactgag
     1801 acccggaatc agttccttga tgagctcatg gagcttgaga tcttcttagc ccagagagca
     1861 gtggagttga gtgaggaggc agatgtcctg tctgtgagcc agttccagct ggctccagcc
     1921 atcctgcagg gccagaccaa agagaagatg gttaccatgg tgtcagtgct ggaggatctg
     1981 attggcaagc ttaccagtct tcagctgcaa cacctgttta tgatcctggc ctcaccaagg
     2041 tatgtggacc gagtgactga attcctccag caaaagctga agcagtccca gctgctggct
     2101 ttgaagaaag agctgatggt gcagaagcag caggaggcac ttgaggagca ggcggctctg
     2161 gagcctaagc tggacctgct actggagaag accaaggagc tgcagaagct gattgaagct
     2221 gacatctcca agaggtacag cgggcgccct gtgaacctga tgggaacctc tctgtgactc
     2281 gagaacaaag cttgaataag cggccgcgac tctagatcat aatcagccat accacatttg
     2341 tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa
     2401 tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca
     2461 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     2521 ccaaactcat caatgtatct taaggcgtaa attgtaagcg ttaatatttt gttaaaattc
     2581 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     2641 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     2701 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     2761 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     2821 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     2881 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     2941 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     3001 gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3061 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3121 tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt cagttagggt
     3181 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3241 cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc
     3301 atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc
     3361 cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg
     3421 ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc
     3481 taggcttttg caaagatcat caagagacag gatgaggatc gtttcgcatg attgaacaag
     3541 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     3601 cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     3661 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa gacgaggcag
     3721 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     3781 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3841 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3901 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3961 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     4021 tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc gaggatctcg
     4081 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     4141 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     4201 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     4261 gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     4321 gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga
     4381 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4441 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accctagggg
     4501 gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta tgacggcaat
     4561 aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg gggttcggtc
     4621 ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt
     4681 ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa
     4741 cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt agattgattt
     4801 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac
     4861 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa
     4921 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc
     4981 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt
     5041 aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg
     5101 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc
     5161 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
     5221 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagtttgga
     5281 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
     5341 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
     5401 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
     5461 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
     5521 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
     5581 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgccatgc attagttatt
     5641 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
     5701 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
     5761 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
     5821 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
     5881 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
     5941 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
     6001 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
     6061 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
     6121 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
     6181 aggtctatat aagcagagct ggtttagtga accgtcagat ccgctagcat tgccacc
//
