LOCUS       sequence-429536-        7817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human CALR ins5 YD/FL-FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7817)
  AUTHORS   Masubuchi N, Araki M, Yang Y, Hayashi E, Imai M, Edahiro Y, Hironaka
            Y, Mizukami Y, Kihara Y, Takei H, Nudejima M, Koike M, Ohsaka A, 
            Komatsu N
  TITLE     Mutant calreticulin interacts with MPL in the secretion pathway for 
            activation on the cell surface.
  JOURNAL   Leukemia. 2020 Feb;34(2):499-509. doi: 10.1038/s41375-019-0564-z. 
            Epub 2019 Aug 28.
  PUBMED    31462733
REFERENCE   2  (bases 1 to 7817)
  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"; doi: 
            "10.1038/s41375-019-0564-z"; journalName: "Leukemia"; date: 
            "2020-02"; volume: "34"; issue: "2"; pages: "499-509"
COMMENT     Sequence Label: pMSCV-IRES-GFP/CALR ins5 YD/FL-FLAG
FEATURES             Location/Qualifiers
     source          1..7817
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    580..921
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             988..1404
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1334..1356
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1372..1388
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             2708..2731
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2778..3327
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2922..2939)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3149..3168
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3330..3332,3333..3335,3336..4049)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(3330..3332,3333..3335,3336..4046)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3375..3396)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3636..3655)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3983..4004
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4211..4725
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4887..4903
                     /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(4911..4917,4918..4935,4936..4941))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4956..4977
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5094..5111)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5265..5853)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5345..5364)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6024..6815,6816..6884))
                     /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     6647..6666
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6885..6989)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7057..7075
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7113..7135)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7235..7254
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7448..7470
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7598..7617
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7817,1..516)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 atgaaagacc ccacctgtag gtttggcaag ctagcttaag taacgccatt ttgcaaggca
       61 tggaaaatac ataactgaga atagagaagt tcagatcaag gttaggaaca gagagacagc
      121 agaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag
      181 aacagatggt ccccagatgc ggtcccgccc tcagcagttt ctagagaacc atcagatgtt
      241 tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt
      301 cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga gcccacaacc
      361 cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg tattcccaat
      421 aaagcctctt gctgtttgca tccgaatcgt ggactcgctg atccttggga gggtctcctc
      481 agattgattg actgcccacc tcgggggtct ttcatttgga ggttccaccg agatttggag
      541 acccctgccc agggaccacc gacccccccg ccgggaggta agctggccag cggtcgtttc
      601 gtgtctgtct ctgtctttgt gcgtgtttgt gccggcatct aatgtttgcg cctgcgtctg
      661 tactagttag ctaactagct ctgtatctgg cggacccgtg gtggaactga cgagttctga
      721 acacccggcc gcaaccctgg gagacgtccc agggactttg ggggccgttt ttgtggcccg
      781 acctgaggaa gggagtcgat gtggaatccg accccgtcag gatatgtggt tctggtagga
      841 gacgagaacc taaaacagtt cccgcctccg tctgaatttt tgctttcggt ttggaaccga
      901 agccgcgcgt cttgtctgct gcagcgctgc agcatcgttc tgtgttgtct ctgtctgact
      961 gtgtttctgt atttgtctga aaattagggc cagactgtta ccactccctt aagtttgacc
     1021 ttaggtcact ggaaagatgt cgagcggatc gctcacaacc agtcggtaga tgtcaagaag
     1081 agacgttggg ttaccttctg ctctgcagaa tggccaacct ttaacgtcgg atggccgcga
     1141 gacggcacct ttaaccgaga cctcatcacc caggttaaga tcaaggtctt ttcacctggc
     1201 ccgcatggac acccagacca ggtcccctac atcgtgacct gggaagcctt ggcttttgac
     1261 ccccctccct gggtcaagcc ctttgtacac cctaagcctc cgcctcctct tcctccatcc
     1321 gccccgtctc tcccccttga acctcctcgt tcgaccccgc ctcgatcctc cctttatcca
     1381 gccctcactc cttctctagg cgccggaatt cgccaccatg ctgctatccg tgccgctgct
     1441 gctcggcctc ctcggcctgg ccgtcgccga gcctgccgtc tacttcaagg agcagtttct
     1501 ggacggagac gggtggactt cccgctggat cgaatccaaa cacaagtcag attttggcaa
     1561 attcgttctc agttccggca agttctacgg tgacgaggag aaagataaag gtttgcagac
     1621 aagccaggat gcacgctttt atgctctgtc ggccagtttc gagcctttca gcaacaaagg
     1681 ccagacgctg gtggtgcagt tcacggtgaa acatgagcag aacatcgact gtgggggcgg
     1741 ctttgtgaag ctgtttccta atagtttgga ccagacagac atgcacggag actcagaata
     1801 caacatcatg tttggtcccc tcatctgtgg ccctggcacc aagaaggttc atgtcatctt
     1861 caactacaag ggcaagaacg tgctgatcaa caaggacatc cgttgcaagg atgatgagtt
     1921 tacacacctg tacacactga ttgtgcggcc agacaacacc tatgaggtga agattgacaa
     1981 cagccaggtg gagtccggct ccttggaaga cgattgggac ttcctgccac ccaagaagat
     2041 aaaggatcct gatgcttcaa aaccggaaga ctgggatgag cgggccaaga tcgatgatcc
     2101 cacagactcc aagcctgagg actgggacaa gcccgagcat atccctgacc ctgatgctaa
     2161 gaagcccgag gactgggatg aagagatgga cggagagtgg gaacccccag tgattcagaa
     2221 ccctgagtac aagggtgagt ggaagccccg gcagatcgac aacccagatt acaagggcac
     2281 ttggatccac ccagaaattg acaaccccga gtattctccc gatcccagta tctatgccta
     2341 tgataacttt ggcgtgctgg gcctggacct ctggcaggtc aagtctggca ccatctttga
     2401 caacttcctc atcaccaacg atgaggcata cgctgaggag tttggcaacg agacgtgggg
     2461 cgtaacaaag gcagcagaga aacaaatgaa ggacaaacag gacgaggagc agaggcttaa
     2521 ggaggaggaa gaagacaaga aacgcaaaga ggaggaggag gcagaggaca attgtcggag
     2581 gatgatgagg acaaagatga ggatgaggag gatgaggagg acaaggagga agatgaggag
     2641 gaagatgtcc ccggccaggc caaggacgag ctgtagagag gcctgcctcc agggctggac
     2701 tgaggccgac tacaaagacg atgacgataa ataggaattc gttaacctcg agcgggatca
     2761 attccgcccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
     2821 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc
     2881 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca
     2941 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac
     3001 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg
     3061 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
     3121 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct
     3181 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg
     3241 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
     3301 ggttttcctt tgaaaaacac gataatacca tggtgagcaa gggcgaggag ctgttcaccg
     3361 gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt
     3421 ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca
     3481 ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac ggcgtgcagt
     3541 gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg
     3601 aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg
     3661 ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact
     3721 tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg
     3781 tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca
     3841 acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg
     3901 acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc ctgagcaaag
     3961 accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca
     4021 ctctcggcat ggacgagctg tacaagtaaa gcggccgcga ctctagagtc gacctgcagg
     4081 catgcaagct tcaggtagcc ggctaacgtt aacaaccggt acctctagaa ctatagctag
     4141 catgcgcaaa tttaaagcgc tgatatcgat aaaataaaag attttattta gtctccagaa
     4201 aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag taacgccatt
     4261 ttgcaaggca tggaaaatac ataactgaga atagagaagt tcagatcaag gttaggaaca
     4321 gagagacagc agaatatggg ccaaacagga tatctgtggt aagcagttcc tgccccggct
     4381 cagggccaag aacagatggt ccccagatgc ggtcccgccc tcagcagttt ctagagaacc
     4441 atcagatgtt tccagggtgc cccaaggacc tgaaatgacc ctgtgcctta tttgaactaa
     4501 ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc tccccgagct caataaaaga
     4561 gcccacaacc cctcactcgg cgcgccagtc ctccgataga ctgcgtcgcc cgggtacccg
     4621 tgtatccaat aaaccctctt gcagttgcat ccgacttgtg gtctcgctgt tccttgggag
     4681 ggtctcctct gagtgattga ctacccgtca gcgggggtct ttcatgggta acagtttctt
     4741 gaagttggag aacaacattc tgagggtagg agtcgaatat taagtaatcc tgactcaatt
     4801 agccactgtt ttgaatccac atactccaat actcctgaaa tagttcatta tggacagcgc
     4861 agaaagagct ggggagaatt gtgaaattgt tatccgctca caattccaca caacatacga
     4921 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     4981 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5041 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     5101 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     5161 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     5221 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     5281 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     5341 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     5401 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     5461 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     5521 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     5581 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5641 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5701 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5761 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5821 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5881 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5941 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     6001 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     6061 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     6121 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     6181 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     6241 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     6301 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     6361 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     6421 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     6481 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     6541 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6601 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6661 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6721 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6781 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6841 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6901 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6961 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     7021 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     7081 cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     7141 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     7201 ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga
     7261 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     7321 cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg
     7381 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca
     7441 gggttttccc agtcacgacg ttgtaaaacg acggcgcaag gaatggtgca tgcaaggaga
     7501 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     7561 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     7621 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggcgat
     7681 tagtccaatt tgttaaagac aggatatcag tggtccaggc tctagttttg actcaacaat
     7741 atcaccagct gaagcctata gagtacgagc catagataaa ataaaagatt ttatttagtc
     7801 tccagaaaaa gggggga
//
