LOCUS       sequence-454102-        7238 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses FLAG tagged human WIPI3..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7238)
  AUTHORS   Shimizu T, Tamura N, Nishimura T, Saito C, Yamamoto H, Mizushima N
  TITLE     Comprehensive analysis of autophagic functions of WIPI family 
            proteins and their implications for the pathogenesis of 
            beta-propeller associated neurodegeneration.
  JOURNAL   Hum Mol Genet. 2023 Aug 7;32(16):2623-2637. doi: 
            10.1093/hmg/ddad096.
  PUBMED    37364041
REFERENCE   2  (bases 1 to 7238)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10"; journalName: 
            "Hum Mol Genet"; date: "2023-08-7- 7"; volume: "32"; issue: "16"; 
            pages: "2623-2637"
COMMENT     Sequence Label: pMRX-IP-hWIPI3-3×FLAG
FEATURES             Location/Qualifiers
     source          1..7238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    739..1096
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1161..1577
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1507..1529
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1545..1561
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    1587..1961
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     1899..1915
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             3050..3115
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    3149..3715
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3165..3717
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3309..3326)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3536..3555
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3735..4334
                     /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(3735..3754)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4231..4251
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4564..5033
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5163..5180)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5334..5922)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5414..5433)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6086..6877,6878..6946))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6709..6728
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6947..7051)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7119..7137
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg ctagagaacc atcagatgtt tccagggtgc cccaaggacc
      421 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
      481 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc
      541 ctccgattga ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat
      601 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca
      661 gcgggggtct ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc
      721 gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg attgtctagt
      781 gtctatgact gattttatgc gcctgcgtcg gtactagtta gctaactagc tctgtatctg
      841 gcggacccgt ggtggaactg acgagttcgg aacacccggc cgcaaccctg ggagacgtcc
      901 cagggacttc gggggccgtt tttgtggccc gacctgagtc caaaaatccc gatcgttttg
      961 gactctttgg tgcacccccc ttagaggagg gatatgtggt tctggtagga gacgagaacc
     1021 taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttgggaccga agccgcgccg
     1081 cgcgtcttgt ctgctgcagc atcgttctgt gttgtctctg tctgactgtg tttctgtatt
     1141 tgtctgaaaa tatgggcccg ggccagactg ttaccactcc cttaagtttg accttaggtc
     1201 actggaaaga tgtcgagcgg atcgctcaca accagtcggt agatgtcaag aagagacgtt
     1261 gggttacctt ctgctctgca gaatggccaa cctttaacgt cggatggccg cgagacggca
     1321 cctttaaccg agacctcatc acccaggtta agatcaaggt cttttcacct ggcccgcatg
     1381 gacacccaga ccaggtcccc tacatcgtga cctgggaagc cttggctttt gacccccctc
     1441 cctgggtcaa gccctttgta caccctaagc ctccgcctcc tcttcctcca tccgccccgt
     1501 ctctccccct tgaacctcct cgttcgaccc cgcctcgatc ctccctttat ccagccctca
     1561 ctccttctct aggcgccccc atatggccat atgagatctt atatggggca cccccgcccc
     1621 ttgtaaactt ccctgaccct gacatgacaa gagttactaa cagcccctct ctccaagctc
     1681 acttacaggc tctctactta gtccagcacg aagtctggag acctctggcg gcagcctacc
     1741 aagaacaact ggaccgaccg gtggtacctc acccttaccg agtcggcgac acagtgtggg
     1801 tccgccgaca ccagactaag aacctagaac ctcgctggaa aggaccttac acagtcctgc
     1861 tgaccacccc caccgccctc aaagtagacg gcatcgcagc ttggatacac gccgcccacg
     1921 tgaaggctgc cgaccccggg ggtggaccat cctctagact gccggatctg ccatcgatat
     1981 aaagcttaaa ggatccatga acctcctgcc gtgtaaccct cacggcaacg ggctgctcta
     2041 cgccggcttc aaccaggacc acggatgctt tgcgtgtggg atggaaaatg gattccgagt
     2101 ctataacact gatccactaa aagaaaaaga gaaacaagaa tttctagaag gaggagttgg
     2161 ccatgttgaa atgttatttc gctgcaacta tttagcttta gttggtggtg gaaaaaagcc
     2221 gaaataccct cccaacaaag taatgatctg ggatgacctg aagaagaaga ctgttattga
     2281 aatagaattt tctacagaag tcaaggcagt caagctgcgg cgagatagaa ttgtggtggt
     2341 tttggactcc atgattaagg tgttcacatt cacacacaat ccccatcagt tgcacgtctt
     2401 cgaaacctgc tataacccca aaggcctctg tgtcctttgt cccaatagta acaactccct
     2461 cctggccttt ccgggcacgc acacgggcca tgtgcagctt gtggacctgg ccagcacgga
     2521 gaagccaccc gtggacattc ctgcacacga gggtgtcctg agctgcattg cactcaacct
     2581 gcagggaaca agaattgcaa ctgcatccga gaaagggacg cttataagaa tatttgatac
     2641 ttcatcaggg catttaatcc aggaactgcg aagaggatct caagcagcca atatttactg
     2701 catcaacttc aatcaggatg cgtccctcat ctgcgtatcc agcgaccacg gcacagtgca
     2761 tatttttgca gctgaagatc caaaaaggaa taaacagtcc agtttggcct cagccagttt
     2821 ccttccaaaa tacttcagtt ccaagtggag tttctccaag tttcaggttc cctcaggctc
     2881 tccgtgcatt tgtgcctttg gaacagagcc aaacgccgtc attgcaattt gtgcagacgg
     2941 cagctactac aaattcctgt tcaaccccaa gggggagtgc atccgagatg tctacgcgca
     3001 gtttctagag atgaccgatg acaagctgga attcggctct ggttcaggcg actacaaaga
     3061 ccatgacggt gattataaag atcatgacat cgactacaag gatgacgatg acaagtaatg
     3121 atttctcgag agcggccgct acgtaaattc cgcccccccc cctaacgtta ctggccgaag
     3181 ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc
     3241 ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg
     3301 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
     3361 tctggaagtt tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc
     3421 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa
     3481 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct
     3541 ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg
     3601 atctgatctg gggcctcggt gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg
     3661 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
     3721 ccacaaccgt cgacatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg
     3781 tcccccgggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca
     3841 ccgtcgaccc ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc
     3901 gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct
     3961 ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg
     4021 ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc
     4081 accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg
     4141 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
     4201 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
     4261 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
     4321 agcccggtgc ctgagtcgac cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg
     4381 cacgacccca tggctcgacg ataaaataaa agattttatt tagtctccag aaaaaggggg
     4441 gaatgaaaga ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg
     4501 catggaaaaa tacataactg agaatagaga agttcagatc aaggtcagga acagatggaa
     4561 cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
     4621 caagaacaga tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct
     4681 gccccggctc agggccaaga acagatggtc cccagatgcg gtccagccct cagcagtttc
     4741 tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat
     4801 ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc
     4861 aataaaagag cccacaaccc ctcactcggc gcgccagtcc tccgattgac tgagtcgccc
     4921 gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt
     4981 ccttgggagg gtctcctctg agtgattgac tacccgtcag cgggggtctt tcacatgcag
     5041 catgtatcaa aattaatttg gttttttttc ttaagtattt acattaaatg gccatagttg
     5101 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     5161 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5221 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5281 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5341 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5401 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5461 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5521 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5581 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5641 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5701 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5761 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5821 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5881 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5941 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     6001 ttatcaaaaa ggatcttcac ctagatcctt ttgcggccgg ccgcaaatca atctaaagta
     6061 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6121 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6181 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6241 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6301 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6361 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6421 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6481 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6541 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6601 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6661 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc
     6721 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6781 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6841 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6901 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6961 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7021 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     7081 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     7141 ttcgtcttca ctataggagc ttggcccatt gcatacgttg tatccatatc ataatatgta
     7201 catttatatt ggctcatgtc caacattacc gccatgtt
//
