LOCUS       sequence-221590-        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"
     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"
     regulatory      1988..1997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1997..2062
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    3328..3894
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    3344..3896
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3488..3505)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3715..3734
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3914..4513
                     /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(3914..3933)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4410..4430
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4743..5212
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5342..5359)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5513..6101)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5593..5612)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6265..7056,7057..7125))
                     /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     6888..6907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7126..7230)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7298..7316
                     /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 aaagcttgcc accatggact acaaagacca tgacggtgat tataaagatc atgacatcga
     2041 ctacaaggat gacgatgaca agggttcagg ctcgggatcc gggaattcga tggcagagaa
     2101 tggaaaaaat tgtgaccaga gacgtgtagc aatgaacaag gaacatcata atggaaattt
     2161 cacagacccc tcttcagtga atgaaaagaa gaggagggag cgggaagaaa ggcagaatat
     2221 tgtcctgtgg agacagccgc tcattacctt gcagtatttt tctctggaaa tccttgtaat
     2281 cttgaaggaa tggacctcaa aattatggca tcgtcaaagc attgtggtgt cttttttact
     2341 gctgcttgct gtgcttatag ctacgtatta tgttgaagga gtgcatcaac agtatgtgca
     2401 acgtatagag aaacagtttc ttttgtatgc ctactggata ggcttaggaa ttttgtcttc
     2461 tgttgggctt ggaacagggc tgcacacctt tctgctttat ctgggtccac atatagcctc
     2521 agttacatta gctgcttatg aatgcaattc agttaatttt cccgaaccac cctatcctga
     2581 tcagattatt tgtccagatg aagagggcac tgaaggaacc atttctttgt ggagtatcat
     2641 ctcaaaagtt aggattgaag cctgcatgtg gggtatcggt acagcaatcg gagagctgcc
     2701 tccatatttc atggccagag cagctcgcct ctcaggtgct gaaccagatg atgaagagta
     2761 tcaggaattt gaagagatgc tggaacatgc agagtctgca caagactttg cctcccgggc
     2821 caaactggca gttcaaaaac tagtacagaa agttggattt tttggaattt tggcctgtgc
     2881 ttcaattcca aatcctttat ttgatctggc tggaataacg tgtggacact ttctggtacc
     2941 tttttggacc ttctttggtg caaccctaat tggaaaagca ataataaaaa tgcatatcca
     3001 gaaaattttt gttataataa cattcagcaa gcacatagtg gagcaaatgg tggctttcat
     3061 tggtgctgtc cccggcatag gtccatctct gcagaagcca tttcaggagt acctggaggc
     3121 tcaacggcag aagcttcacc acaaaagcga aatgggcaca ccacagggag aaaactggtt
     3181 gtcctggatg tttgaaaagt tggtcgttgt catggtgtgt tacttcatcc tatctatcat
     3241 taactccatg gcacaaagtt atgccaaacg aatccagcag cggttgaact cagaggagaa
     3301 aactaaataa gcggccgcta cgtaaattcc gccccccccc ctaacgttac tggccgaagc
     3361 cgcttggaat aaggccggtg tgcgtttgtc tatatgttat tttccaccat attgccgtct
     3421 tttggcaatg tgagggcccg gaaacctggc cctgtcttct tgacgagcat tcctaggggt
     3481 ctttcccctc tcgccaaagg aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct
     3541 ctggaagttt cttgaagaca aacaacgtct gtagcgaccc tttgcaggca gcggaacccc
     3601 ccacctggcg acaggtgcct ctgcggccaa aagccacgtg tataagatac acctgcaaag
     3661 gcggcacaac cccagtgcca cgttgtgagt tggatagttg tggaaagagt caaatggctc
     3721 tcctcaagcg tattcaacaa ggggctgaag gatgcccaga aggtacccca ttgtatggga
     3781 tctgatctgg ggcctcggtg cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt
     3841 ctaggccccc cgaaccacgg ggacgtggtt ttcctttgaa aaacacgatg ataatatggc
     3901 cacaaccgtc gacatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     3961 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     4021 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     4081 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     4141 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     4201 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     4261 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     4321 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     4381 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     4441 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     4501 gcccggtgcc tgagtcgacc gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc
     4561 acgaccccat ggctcgacga taaaataaaa gattttattt agtctccaga aaaagggggg
     4621 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
     4681 atggaaaaat acataactga gaatagagaa gttcagatca aggtcaggaa cagatggaac
     4741 agctgaatat gggccaaaca ggatatctgt ggtaagcagt tcctgccccg gctcagggcc
     4801 aagaacagat ggaacagctg aatatgggcc aaacaggata tctgtggtaa gcagttcctg
     4861 ccccggctca gggccaagaa cagatggtcc ccagatgcgg tccagccctc agcagtttct
     4921 agagaaccat cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt
     4981 tgaactaacc aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca
     5041 ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg
     5101 ggtacccgtg tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc
     5161 cttgggaggg tctcctctga gtgattgact acccgtcagc gggggtcttt cacatgcagc
     5221 atgtatcaaa attaatttgg ttttttttct taagtattta cattaaatgg ccatagttgc
     5281 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5341 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5401 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5461 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5521 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5581 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5641 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5701 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5761 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5821 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5881 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5941 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6001 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6061 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6121 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6181 tatcaaaaag gatcttcacc tagatccttt tgcggccggc cgcaaatcaa tctaaagtat
     6241 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6301 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6361 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6421 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6481 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6541 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6601 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6661 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6721 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6781 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6841 atagtgtatg cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc
     6901 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6961 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     7021 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     7081 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     7141 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     7201 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7261 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     7321 tcgtcttcac tataggagct tggcccattg catacgttgt atccatatca taatatgtac
     7381 atttatattg gctcatgtcc aacattaccg ccatgtt
//
