LOCUS       sequence-403935-        6646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3xFLAG-tagged UFSP2 C302A in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6646)
  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 6646)
  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-UFSP2 C302A
FEATURES             Location/Qualifiers
     source          1..6646
                     /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"
     intron          2533..2762
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     primer_bind     2745..2764
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     misc_feature    2820..3393
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2987..3004)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3214..3233
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3426..4025
                     /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(3426..3445)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3922..3942
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4263..4384
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4300..4319)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(4489..4505)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4489..4505)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4502..4524)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4513..4529
                     /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(4537..4543,4544..4561,4562..4567))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4582..4603
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4720..4737)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4891..5479)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4971..4990)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5650..6441,6442..6510))
                     /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     6273..6292
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6511..6615)
                     /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 caaggaattc atggtgattt cagaaagtat ggatatactc
     1081 ttcagaataa gaggaggcct tgatttggct tttcagctag ctactcctaa tgaaattttt
     1141 ctcaagaagg cactgaaaca tgtgttgagt gacctgtcaa ctaagctgtc ttcaaacgcc
     1201 cttgtgttca gaatttgcca cagttcagtg tatatatggc ctagcagtga cataaacacc
     1261 attcctggag aactgactga tgcttctgct tgtaagaaca tactgcgctt tattcaattt
     1321 gagccagaag aagatataaa aagaaaattc atgagaaaga aggacaaaaa gttatcagac
     1381 atgcatcaaa tagtaaatat agatcttatg ctggaaatgt caacctccct ggcagctgta
     1441 acgcccatca ttgaaaggga aagcggagga caccattatg ttaatatgac tttacctgtc
     1501 gatgcagtta tatctgttgc tccagaagaa acatggggaa aagttcgtaa actcctggtt
     1561 gatgcaattc ataatcaact aactgacatg gaaaaatgta ttttgaaata tatgaaagga
     1621 acatctattg tggtccctga accactgcac tttttattac cagggaaaaa aaatcttgta
     1681 acaatttcat atccttcagg aataccagat ggccagctgc aggcctatag gaaggagtta
     1741 catgatcttt tcaatctgcc tcacgacaga ccctatttca aaaggtctaa tgcttatcac
     1801 tttccagatg agccatacaa agatggttac attagaaatc cacatactta ccttaatcca
     1861 cctaacatgg agactggtat gatttatgtg gtccagggca tatatggcta tcatcattat
     1921 atgcaggatc gcatagatga caatggctgg ggcgctgctt atcgatctct gcagactatc
     1981 tgctcttggt tcaaacatca gggatacaca gagaggtcca ttccaacaca cagagaaatt
     2041 cagcaggctc tagtcgatgc cggggacaaa ccagcaacat ttgtcggatc gcggcaatgg
     2101 attggatcta ttgaggtgca gctggtacta aaccaattga tcggtataac gtcaaaaatc
     2161 ctgtttgtca gccaaggttc agaaattgcc tctcaaggac gggaactggc taatcatttc
     2221 caaagtgaag gaactccagt tatgatcggg ggaggagttt tggcccacac aatactagga
     2281 gttgcatgga atgagattac agggcagata aagtttctga ttctagatcc acattatacc
     2341 ggtgctgaag acctgcaagt tattttggaa aagggctggt gcggatggaa gggcccagat
     2401 ttttggaaca aggatgcata ctataactta tgtcttcctc agcgaccaaa tatgatttaa
     2461 ctcgagggcc gcatagataa ctgatccagt gtgctggaat taattcgctg tctgcgaggg
     2521 ccagctgttg gggtgagtac tccctctcaa aagcgggcat gacttctgcg ctaagattgt
     2581 cagtttccaa aaacgaggag gatttgatat tcacctggcc cgcggtgatg cctttgaggg
     2641 tggccgcgtc catctggtca gaaaagacaa tctttttgtt gtcaagcttg aggtgtggca
     2701 ggcttgagat ctggccatac acttgagtga caatgacatc cactttgcct ttctctccac
     2761 aggtgtccac tcccaggtcc aactgcaggt cgagcatgca tctagggcgg ccaattccgc
     2821 ccctctcccc cccccccccc taacgttact ggccgaagcc gcttggaata aggccggtgt
     2881 gcgtttgtct atatgttatt ttccaccata ttgccgtctt ttggcaatgt gagggcccgg
     2941 aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct cgccaaagga
     3001 atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa
     3061 acaacgtctg tagcgaccct ttgcaggcag cggaaccccc cacctggcga caggtgcctc
     3121 tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc ccagtgccac
     3181 gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt attcaacaag
     3241 gggctgaagg atgcccagaa ggtaccccat tgtatgggat ctgatctggg gcctcggtgc
     3301 acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg
     3361 gacgtggttt tcctttgaaa aacacgatga taagcttgcc acaacccaca aggagacgac
     3421 cttccatgac cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtcccccggg
     3481 ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgacc
     3541 cggaccgcca catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc
     3601 tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc
     3661 cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga
     3721 gcggttcccg gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca
     3781 aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc
     3841 tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct
     3901 tcctggagac ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca
     3961 ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg
     4021 cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag gagcgcacga ccccatggct
     4081 ccgaccgaag ccacccgggg cggccccgcc gaccccgcac ccgcccccga ggcccaccga
     4141 ctctagctag ataactgatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt
     4201 taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
     4261 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4321 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4381 cttaacgctc gatcgagtgc attctagttg tggtttgtcc aaactcatca atgtatctta
     4441 tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     4501 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4561 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4621 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4681 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4741 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4801 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4861 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4921 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4981 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5041 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5101 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5161 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5221 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5281 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5341 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5401 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5461 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     5521 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     5581 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     5641 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5701 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5761 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5821 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5881 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5941 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6001 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6061 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6121 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6181 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6241 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6301 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6361 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6421 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6481 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     6541 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     6601 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
