LOCUS       sequence-434056-        7544 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses FLAG tagged human WIPI1A..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7544)
  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 7544)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-hWIPI1A-3×FLAG
FEATURES             Location/Qualifiers
     source          1..7544
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     91..111
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             424..893
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(1023..1040)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1194..1782)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1274..1293)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1946..2737,2738..2806))
                     /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     2569..2588
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2807..2911)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2979..2997
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        3099..3478
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    3837..4194
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             4259..4675
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     4605..4627
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     4643..4659
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    4685..5059
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     4997..5013
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     CDS             6454..6519
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    6553..7119
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    6569..7121
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(6713..6730)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     6940..6959
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(7139..7544,1..194)
                     /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(7139..7158)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
ORIGIN
        1 gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg
       61 tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct
      121 tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca
      181 agcccggtgc ctgagtcgac cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg
      241 cacgacccca tggctcgacg ataaaataaa agattttatt tagtctccag aaaaaggggg
      301 gaatgaaaga ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg
      361 catggaaaaa tacataactg agaatagaga agttcagatc aaggtcagga acagatggaa
      421 cagctgaata tgggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcagggc
      481 caagaacaga tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct
      541 gccccggctc agggccaaga acagatggtc cccagatgcg gtccagccct cagcagtttc
      601 tagagaacca tcagatgttt ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat
      661 ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc
      721 aataaaagag cccacaaccc ctcactcggg gcgccagtcc tccgattgac tgagtcgccc
      781 gggtacccgt gtatccaata aaccctcttg cagttgcatc cgacttgtgg tctcgctgtt
      841 ccttgggagg gtctcctctg agtgattgac tacccgtcag cgggggtctt tcacatgcag
      901 catgtatcaa aattaatttg gttttttttc ttaagtattt acattaaatg gccatagttg
      961 cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct
     1021 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     1081 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     1141 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     1201 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1261 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1321 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1381 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1441 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1501 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1561 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1621 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1681 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1741 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     1801 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     1861 ttatcaaaaa ggatcttcac ctagatcctt ttgcggccgg ccgcaaatca atctaaagta
     1921 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     1981 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     2041 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     2101 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     2161 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     2221 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     2281 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     2341 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     2401 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     2461 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     2521 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc
     2581 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     2641 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     2701 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     2761 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     2821 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     2881 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     2941 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     3001 ttcgtcttca ctataggagc ttggcccatt gcatacgttg tatccatatc ataatatgta
     3061 catttatatt ggctcatgtc caacattacc gccatgttga cattgattat tgactagtta
     3121 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     3181 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     3241 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     3301 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
     3361 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
     3421 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatgct
     3481 agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt
     3541 tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca
     3601 ataaaagagc ccacaacccc tcactcggcg cgccagtcct ccgattgact gagtcgcccg
     3661 ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc
     3721 cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt catttggggg
     3781 ctcgtccggg atcgggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc
     3841 tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgactga ttttatgcgc
     3901 ctgcgtcggt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac
     3961 gagttcggaa cacccggccg caaccctggg agacgtccca gggacttcgg gggccgtttt
     4021 tgtggcccga cctgagtcca aaaatcccga tcgttttgga ctctttggtg cacccccctt
     4081 agaggaggga tatgtggttc tggtaggaga cgagaaccta aaacagttcc cgcctccgtc
     4141 tgaatttttg ctttcggttt gggaccgaag ccgcgccgcg cgtcttgtct gctgcagcat
     4201 cgttctgtgt tgtctctgtc tgactgtgtt tctgtatttg tctgaaaata tgggcccggg
     4261 ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg tcgagcggat
     4321 cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct gctctgcaga
     4381 atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag acctcatcac
     4441 ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc aggtccccta
     4501 catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc cctttgtaca
     4561 ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg aacctcctcg
     4621 ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag gcgcccccat
     4681 atggccatat gagatcttat atggggcacc cccgcccctt gtaaacttcc ctgaccctga
     4741 catgacaaga gttactaaca gcccctctct ccaagctcac ttacaggctc tctacttagt
     4801 ccagcacgaa gtctggagac ctctggcggc agcctaccaa gaacaactgg accgaccggt
     4861 ggtacctcac ccttaccgag tcggcgacac agtgtgggtc cgccgacacc agactaagaa
     4921 cctagaacct cgctggaaag gaccttacac agtcctgctg accaccccca ccgccctcaa
     4981 agtagacggc atcgcagctt ggatacacgc cgcccacgtg aaggctgccg accccggggg
     5041 tggaccatcc tctagactgc cggatctgcc atcgatataa agcttaaagg atccatggag
     5101 gccgaggccg cggacgctcc cccgggcggg gttgagtcgg cgctcagctg cttctctttc
     5161 aaccaggact gcacatccct agcaactgga actaaagccg ggtataagct gttttctctg
     5221 agttctgtgg agcagctgga tcaagtccac ggaagcaatg aaatcccgga cgtctacatc
     5281 gtggagcgcc tcttctccag cagcctggtg gtggtagtca gtcacacaaa accacggcag
     5341 atgaacgtgt atcacttcaa gaaaggcaca gagatctgta attacagcta ctccagcaac
     5401 atcttgtcca taaggctgaa ccggcaaagg ctgctggttt gcctagaaga gtccatttat
     5461 attcacaaca ttaaagacat gaagctgttg aagaccctcc tggatattcc tgcaaaccca
     5521 acaggtctat gtgctctctc tatcaaccat tccaattctt acctggccta tcctggaagc
     5581 ctgacttcag gggagattgt gctttatgat ggaaactccc tgaaaacagt ctgcactatt
     5641 gctgcccatg agggaacact agctgccatc accttcaatg cctcaggctc caaactagca
     5701 agtgcgtctg aaaaaggcac agtcatccgg gtgttctctg tccctgatgg gcaaaagctc
     5761 tatgagttcc ggagagggat gaaaaggtat gtgacaatca gctctctagt gttcagtatg
     5821 gattcacaat tcctctgcgc ctccagtaac accgagacgg tacacatctt caagctggaa
     5881 caggtcacca acagtcgacc agaagagcct tcgacctgga gtggctacat gggaaagatg
     5941 tttatggctg ctaccaacta cctccctacc caggtgtcag acatgatgca tcaggacagg
     6001 gcttttgcca ctgcacgctt gaacttctcc ggacagagga acatctgtac cctctcaacg
     6061 atccagaagt tgccacggct gctagttgcg tcatccagtg gacaccttta tatgtacaat
     6121 ttggatcctc aggatggagg agagtgtgtc ttaatcaaaa cccacagctt gcttggctca
     6181 ggaacaacag aagagaataa agaaaatgac ctcagacctt ccttacctca gtcttatgca
     6241 gcgaccgtag ccagaccaag tgcatcttca gcctccacgg tgccaggtta ttctgaggac
     6301 ggcggggcgc tgcgaggaga agttattcct gaacatgagt ttgcgacggg accagtgtgt
     6361 cttgatgatg agaatgagtt tcctcctata atcttgtgcc gtggaaatca gaagggcaaa
     6421 acgaagcagt cagaattcgg ctctggttca ggcgactaca aagaccatga cggtgattat
     6481 aaagatcatg acatcgacta caaggatgac gatgacaagt aatgatttct cgagagcggc
     6541 cgctacgtaa attccgcccc cccccctaac gttactggcc gaagccgctt ggaataaggc
     6601 cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg
     6661 gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc
     6721 aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agtttcttga
     6781 agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg
     6841 tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag
     6901 tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc
     6961 aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct
     7021 cggtgcacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac
     7081 cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa ccgtcgacat
     7141 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg
     7201 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg
     7261 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     7321 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     7381 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     7441 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     7501 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagg
//
