LOCUS       sequence-224938-        7417 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses VMP1 tagged with 3xFLAG in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7417)
  AUTHORS   Morita K, Hama Y, Izume T, Tamura N, Ueno T, Yamashita Y, Sakamaki 
            Y, Mimura K, Morishita H, Shihoya W, Nureki O, Mano H, Mizushima N
  TITLE     Genome-wide CRISPR screen identifies TMEM41B as a gene required for 
            autophagosome formation.
  JOURNAL   J Cell Biol. 2018 Aug 9. pii: jcb.201804132. doi: 
            10.1083/jcb.201804132.
  PUBMED    30093494
REFERENCE   2  (bases 1 to 7417)
  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: "J Cell 
            Biol. 2018 Aug 9. pii: jcb.201804132. doi: 10.1083/jcb.201804132."
COMMENT     Sequence Label: pMRXIP 3xFLAG-VMP1
FEATURES             Location/Qualifiers
     source          1..7417
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    1266..1832
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    1282..1834
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1426..1443)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1653..1672
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             1852..2451
                     /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(1852..1871)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2348..2368
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2681..3150
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(3280..3297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3451..4039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3531..3550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4203..4994,4995..5063))
                     /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     4826..4845
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5064..5168)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5236..5254
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        5356..5735
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    6094..6451
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6516..6932
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6862..6884
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6900..6916
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     misc_feature    6942..7316
                     /label=pol region
                     /note="Moloney murine leukemia virus (MMLV) pol region 
                     containing the splice acceptor site"
     primer_bind     7254..7270
                     /label=pBMN 5'
                     /note="MMLV sequence, for inserts in pBMN retroviral 
                     vector"
     regulatory      7343..7352
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7352..7417
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
ORIGIN
        1 ggttcaggct cgggatccgg gaattcgatg gcagagaatg gaaaaaattg tgaccagaga
       61 cgtgtagcaa tgaacaagga acatcataat ggaaatttca cagacccctc ttcagtgaat
      121 gaaaagaaga ggagggagcg ggaagaaagg cagaatattg tcctgtggag acagccgctc
      181 attaccttgc agtatttttc tctggaaatc cttgtaatct tgaaggaatg gacctcaaaa
      241 ttatggcatc gtcaaagcat tgtggtgtct tttttactgc tgcttgctgt gcttatagct
      301 acgtattatg ttgaaggagt gcatcaacag tatgtgcaac gtatagagaa acagtttctt
      361 ttgtatgcct actggatagg cttaggaatt ttgtcttctg ttgggcttgg aacagggctg
      421 cacacctttc tgctttatct gggtccacat atagcctcag ttacattagc tgcttatgaa
      481 tgcaattcag ttaattttcc cgaaccaccc tatcctgatc agattatttg tccagatgaa
      541 gagggcactg aaggaaccat ttctttgtgg agtatcatct caaaagttag gattgaagcc
      601 tgcatgtggg gtatcggtac agcaatcgga gagctgcctc catatttcat ggccagagca
      661 gctcgcctct caggtgctga accagatgat gaagagtatc aggaatttga agagatgctg
      721 gaacatgcag agtctgcaca agactttgcc tcccgggcca aactggcagt tcaaaaacta
      781 gtacagaaag ttggattttt tggaattttg gcctgtgctt caattccaaa tcctttattt
      841 gatctggctg gaataacgtg tggacacttt ctggtacctt tttggacctt ctttggtgca
      901 accctaattg gaaaagcaat aataaaaatg catatccaga aaatttttgt tataataaca
      961 ttcagcaagc acatagtgga gcaaatggtg gctttcattg gtgctgtccc cggcataggt
     1021 ccatctctgc agaagccatt tcaggagtac ctggaggctc aacggcagaa gcttcaccac
     1081 aaaagcgaaa tgggcacacc acagggagaa aactggttgt cctggatgtt tgaaaagttg
     1141 gtcgttgtca tggtgtgtta cttcatccta tctatcatta actccatggc acaaagttat
     1201 gccaaacgaa tccagcagcg gttgaactca gaggagaaaa ctaaataagc ggccgctacg
     1261 taaattccgc ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg
     1321 cgtttgtcta tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga
     1381 aacctggccc tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa
     1441 tgcaaggtct gttgaatgtc gtgaaggaag cagttcctct ggaagtttct tgaagacaaa
     1501 caacgtctgt agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct
     1561 gcggccaaaa gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg
     1621 ttgtgagttg gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg
     1681 ggctgaagga tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca
     1741 catgctttac atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg
     1801 acgtggtttt cctttgaaaa acacgatgat aatatggcca caaccgtcga catgaccgag
     1861 tacaagccca cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt acgcaccctc
     1921 gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgacccgga ccgccacatc
     1981 gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag
     2041 gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa
     2101 gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg
     2161 gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg
     2221 ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc
     2281 gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc
     2341 gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag
     2401 gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg agtcgaccgc
     2461 ccgccccacg acccgcagcg cccgaccgaa aggagcgcac gaccccatgg ctcgacgata
     2521 aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc cacctgtagg
     2581 tttggcaagc tagcttaagt aacgccattt tgcaaggcat ggaaaaatac ataactgaga
     2641 atagagaagt tcagatcaag gtcaggaaca gatggaacag ctgaatatgg gccaaacagg
     2701 atatctgtgg taagcagttc ctgccccggc tcagggccaa gaacagatgg aacagctgaa
     2761 tatgggccaa acaggatatc tgtggtaagc agttcctgcc ccggctcagg gccaagaaca
     2821 gatggtcccc agatgcggtc cagccctcag cagtttctag agaaccatca gatgtttcca
     2881 gggtgcccca aggacctgaa atgaccctgt gccttatttg aactaaccaa tcagttcgct
     2941 tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat aaaagagccc acaacccctc
     3001 actcggggcg ccagtcctcc gattgactga gtcgcccggg tacccgtgta tccaataaac
     3061 cctcttgcag ttgcatccga cttgtggtct cgctgttcct tgggagggtc tcctctgagt
     3121 gattgactac ccgtcagcgg gggtctttca catgcagcat gtatcaaaat taatttggtt
     3181 ttttttctta agtatttaca ttaaatggcc atagttgcat taatgaatcg gccaacgcgc
     3241 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     3301 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     3361 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     3421 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     3481 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     3541 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     3601 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     3661 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     3721 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     3781 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3841 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3901 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3961 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4021 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4081 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4141 gatccttttg cggccggccg caaatcaatc taaagtatat atgagtaaac ttggtctgac
     4201 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     4261 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     4321 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     4381 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     4441 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     4501 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     4561 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     4621 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     4681 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     4741 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     4801 tgctcttgcc cggcgtcaac acgggataat accgcgccac atagcagaac tttaaaagtg
     4861 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     4921 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     4981 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     5041 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     5101 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     5161 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     5221 acattaacct ataaaaatag gcgtatcacg aggccctttc gtcttcacta taggagcttg
     5281 gcccattgca tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa
     5341 cattaccgcc atgttgacat tgattattga ctagttatta atagtaatca attacggggt
     5401 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     5461 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     5521 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     5581 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     5641 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     5701 agtacatcta cgtattagtc atcgctatta ccatgctaga gaaccatcag atgtttccag
     5761 ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt
     5821 ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca caacccctca
     5881 ctcggcgcgc cagtcctccg attgactgag tcgcccgggt acccgtgtat ccaataaacc
     5941 ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct cctctgagtg
     6001 attgactacc cgtcagcggg ggtctttcat ttgggggctc gtccgggatc gggagacccc
     6061 tgcccaggga ccaccgaccc accaccggga ggtaagctgg ccagcaactt atctgtgtct
     6121 gtccgattgt ctagtgtcta tgactgattt tatgcgcctg cgtcggtact agttagctaa
     6181 ctagctctgt atctggcgga cccgtggtgg aactgacgag ttcggaacac ccggccgcaa
     6241 ccctgggaga cgtcccaggg acttcggggg ccgtttttgt ggcccgacct gagtccaaaa
     6301 atcccgatcg ttttggactc tttggtgcac cccccttaga ggagggatat gtggttctgg
     6361 taggagacga gaacctaaaa cagttcccgc ctccgtctga atttttgctt tcggtttggg
     6421 accgaagccg cgccgcgcgt cttgtctgct gcagcatcgt tctgtgttgt ctctgtctga
     6481 ctgtgtttct gtatttgtct gaaaatatgg gcccgggcca gactgttacc actcccttaa
     6541 gtttgacctt aggtcactgg aaagatgtcg agcggatcgc tcacaaccag tcggtagatg
     6601 tcaagaagag acgttgggtt accttctgct ctgcagaatg gccaaccttt aacgtcggat
     6661 ggccgcgaga cggcaccttt aaccgagacc tcatcaccca ggttaagatc aaggtctttt
     6721 cacctggccc gcatggacac ccagaccagg tcccctacat cgtgacctgg gaagccttgg
     6781 cttttgaccc ccctccctgg gtcaagccct ttgtacaccc taagcctccg cctcctcttc
     6841 ctccatccgc cccgtctctc ccccttgaac ctcctcgttc gaccccgcct cgatcctccc
     6901 tttatccagc cctcactcct tctctaggcg cccccatatg gccatatgag atcttatatg
     6961 gggcaccccc gccccttgta aacttccctg accctgacat gacaagagtt actaacagcc
     7021 cctctctcca agctcactta caggctctct acttagtcca gcacgaagtc tggagacctc
     7081 tggcggcagc ctaccaagaa caactggacc gaccggtggt acctcaccct taccgagtcg
     7141 gcgacacagt gtgggtccgc cgacaccaga ctaagaacct agaacctcgc tggaaaggac
     7201 cttacacagt cctgctgacc acccccaccg ccctcaaagt agacggcatc gcagcttgga
     7261 tacacgccgc ccacgtgaag gctgccgacc ccgggggtgg accatcctct agactgccgg
     7321 atctgccatc gatataaagc ttgccaccat ggactacaaa gaccatgacg gtgattataa
     7381 agatcatgac atcgactaca aggatgacga tgacaag
//
