LOCUS       sequence-417226-        7778 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of human CALR wt-FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7778)
  AUTHORS   Araki M, Yang Y, Masubuchi N, Hironaka Y, Takei H, Morishita S, 
            Mizukami Y, Kan S, Shirane S, Edahiro Y, Sunami Y, Ohsaka A, Komatsu
            N
  TITLE     Activation of the thrombopoietin receptor by mutant calreticulin in 
            CALR-mutant myeloproliferative neoplasms.
  JOURNAL   Blood. 2016 Jan 27. pii: blood-2015-09-671172.
  PUBMED    26817954
REFERENCE   2  (bases 1 to 7778)
  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: "Blood. 2016
            Jan 27. pii: blood-2015-09-671172."
COMMENT     Sequence Label: pMSCV-IRES-GFP/CALR wt-FLAG
FEATURES             Location/Qualifiers
     source          1..7778
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..517
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    581..922
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             989..1405
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1335..1357
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1373..1389
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     CDS             2670..2693
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2740..3289
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2884..2901)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3111..3130
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3292..3294,3295..3297,3298..4011)
                     /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(3292..3294,3295..3297,3298..4008)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3337..3358)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3598..3617)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3945..3966
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4173..4687
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4849..4865
                     /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(4873..4879,4880..4897,4898..4903))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4918..4939
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5056..5073)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5227..5815)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5307..5326)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5986..6777,6778..6846))
                     /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     6609..6628
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6847..6951)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7019..7037
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7075..7097)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7197..7216
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7410..7432
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7560..7579
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
      121 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
      241 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtattcccaa
      421 taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct
      481 cagattgatt gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga
      541 gacccctgcc cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt
      601 cgtgtctgtc tctgtctttg tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct
      661 gtactagtta gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg
      721 aacacccggc cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc
      781 gacctgagga agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg
      841 agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg
      901 aagccgcgcg tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac
      961 tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac
     1021 cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa
     1081 gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg
     1141 agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg
     1201 cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga
     1261 cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc
     1321 cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc
     1381 agccctcact ccttctctag gcgccggaat tcgccaccat gctgctatcc gtgccgctgc
     1441 tgctcggcct cctcggcctg gccgtcgccg agcctgccgt ctacttcaag gagcagtttc
     1501 tggacggaga cgggtggact tcccgctgga tcgaatccaa acacaagtca gattttggca
     1561 aattcgttct cagttccggc aagttctacg gtgacgagga gaaagataaa ggtttgcaga
     1621 caagccagga tgcacgcttt tatgctctgt cggccagttt cgagcctttc agcaacaaag
     1681 gccagacgct ggtggtgcag ttcacggtga aacatgagca gaacatcgac tgtgggggcg
     1741 gctatgtgaa gctgtttcct aatagtttgg accagacaga catgcacgga gactcagaat
     1801 acaacatcat gtttggtccc gacatctgtg gccctggcac caagaaggtt catgtcatct
     1861 tcaactacaa gggcaagaac gtgctgatca acaaggacat ccgttgcaag gatgatgagt
     1921 ttacacacct gtacacactg attgtgcggc cagacaacac ctatgaggtg aagattgaca
     1981 acagccaggt ggagtccggc tccttggaag acgattggga cttcctgcca cccaagaaga
     2041 taaaggatcc tgatgcttca aaaccggaag actgggatga gcgggccaag atcgatgatc
     2101 ccacagactc caagcctgag gactgggaca agcccgagca tatccctgac cctgatgcta
     2161 agaagcccga ggactgggat gaagagatgg acggagagtg ggaaccccca gtgattcaga
     2221 accctgagta caagggtgag tggaagcccc ggcagatcga caacccagat tacaagggca
     2281 cttggatcca cccagaaatt gacaaccccg agtattctcc cgatcccagt atctatgcct
     2341 atgataactt tggcgtgctg ggcctggacc tctggcaggt caagtctggc accatctttg
     2401 acaacttcct catcaccaac gatgaggcat acgctgagga gtttggcaac gagacgtggg
     2461 gcgtaacaaa ggcagcagag aaacaaatga aggacaaaca ggacgaggag cagaggctta
     2521 aggaggagga agaagacaag aaacgcaaag aggaggagga ggcagaggac aaggaggatg
     2581 atgaggacaa agatgaggat gaggaggatg aggaggacaa ggaggaagat gaggaggaag
     2641 atgtcccggg ccaggccaag gacgagctgg actacaaaga cgatgacgat aaataggaat
     2701 tcgttaacct cgagcgggat caattccgcc ccccccctaa cgttactggc cgaagccgct
     2761 tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg
     2821 gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt
     2881 cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg
     2941 aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac
     3001 ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg
     3061 cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct
     3121 caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg
     3181 atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag
     3241 gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgataatac catggtgagc
     3301 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     3361 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     3421 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     3481 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     3541 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     3601 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     3661 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     3721 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     3781 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     3841 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3901 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3961 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aagcggccgc
     4021 gactctagag tcgacctgca ggcatgcaag cttcaggtag ccggctaacg ttaacaaccg
     4081 gtacctctag aactatagct agcatgcgca aatttaaagc gctgatatcg ataaaataaa
     4141 agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca
     4201 agctagctta agtaacgcca ttttgcaagg catggaaaat acataactga gaatagagaa
     4261 gttcagatca aggttaggaa cagagagaca gcagaatatg ggccaaacag gatatctgtg
     4321 gtaagcagtt cctgccccgg ctcagggcca agaacagatg gtccccagat gcggtcccgc
     4381 cctcagcagt ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga
     4441 ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct
     4501 gctccccgag ctcaataaaa gagcccacaa cccctcactc ggcgcgccag tcctccgata
     4561 gactgcgtcg cccgggtacc cgtgtatcca ataaaccctc ttgcagttgc atccgacttg
     4621 tggtctcgct gttccttggg agggtctcct ctgagtgatt gactacccgt cagcgggggt
     4681 ctttcatggg taacagtttc ttgaagttgg agaacaacat tctgagggta ggagtcgaat
     4741 attaagtaat cctgactcaa ttagccactg ttttgaatcc acatactcca atactcctga
     4801 aatagttcat tatggacagc gcagaaagag ctggggagaa ttgtgaaatt gttatccgct
     4861 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4921 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4981 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     5041 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     5101 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5161 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5221 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5281 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5341 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5401 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5461 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5521 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5581 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5641 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     5701 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5761 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5821 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5881 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5941 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     6001 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     6061 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6121 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6181 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6241 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6301 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6361 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6421 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6481 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6541 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6601 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6661 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6721 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6781 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6841 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6901 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6961 ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa
     7021 taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg
     7081 acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
     7141 agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc
     7201 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     7261 aaggagaaaa taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg
     7321 gcgatcggtg cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag
     7381 gcgattaagt tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggcgca
     7441 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     7501 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     7561 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     7621 atgcgtccgg cgtagaggcg attagtccaa tttgttaaag acaggatatc agtggtccag
     7681 gctctagttt tgactcaaca atatcaccag ctgaagccta tagagtacga gccatagata
     7741 aaataaaaga ttttatttag tctccagaaa aagggggg
//
