LOCUS       sequence-407209-        7817 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral expression of human CALR ins5-FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7817)
  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 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"; journalName: "Blood. 2016
            Jan 27. pii: blood-2015-09-671172."
COMMENT     Sequence Label: pMSCV-IRES-GFP/CALR ins5-FLAG
FEATURES             Location/Qualifiers
     source          1..7817
                     /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             2709..2732
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    2779..3328
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2923..2940)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3150..3169
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3331..3333,3334..3336,3337..4050)
                     /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(3331..3333,3334..3336,3337..4047)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3376..3397)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3637..3656)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3984..4005
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4212..4726
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4888..4904
                     /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(4912..4918,4919..4936,4937..4942))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4957..4978
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5095..5112)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5266..5854)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5346..5365)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6025..6816,6817..6885))
                     /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     6648..6667
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6886..6990)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7058..7076
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7114..7136)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7236..7255
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7449..7471
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7599..7618
                     /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 aattgtcgga
     2581 ggatgatgag gacaaagatg aggatgagga ggatgaggag gacaaggagg aagatgagga
     2641 ggaagatgtc cccggccagg ccaaggacga gctgtagaga ggcctgcctc cagggctgga
     2701 ctgaggccga ctacaaagac gatgacgata aataggaatt cgttaacctc gagcgggatc
     2761 aattccgccc cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt
     2821 ttgtctatat gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac
     2881 ctggccctgt cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc
     2941 aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa
     3001 cgtctgtagc gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg
     3061 gccaaaagcc acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg
     3121 tgagttggat agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc
     3181 tgaaggatgc ccagaaggta ccccattgta tgggatctga tctggggcct cggtgcacat
     3241 gctttacatg tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg
     3301 tggttttcct ttgaaaaaca cgataatacc atggtgagca agggcgagga gctgttcacc
     3361 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     3421 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     3481 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
     3541 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     3601 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     3661 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     3721 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
     3781 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     3841 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     3901 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     3961 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     4021 actctcggca tggacgagct gtacaagtaa agcggccgcg actctagagt cgacctgcag
     4081 gcatgcaagc ttcaggtagc cggctaacgt taacaaccgg tacctctaga actatagcta
     4141 gcatgcgcaa atttaaagcg ctgatatcga taaaataaaa gattttattt agtctccaga
     4201 aaaagggggg aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat
     4261 tttgcaaggc atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac
     4321 agagagacag cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc
     4381 tcagggccaa gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac
     4441 catcagatgt ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta
     4501 accaatcagt tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag
     4561 agcccacaac ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc
     4621 gtgtatccaa taaaccctct tgcagttgca tccgacttgt ggtctcgctg ttccttggga
     4681 gggtctcctc tgagtgattg actacccgtc agcgggggtc tttcatgggt aacagtttct
     4741 tgaagttgga gaacaacatt ctgagggtag gagtcgaata ttaagtaatc ctgactcaat
     4801 tagccactgt tttgaatcca catactccaa tactcctgaa atagttcatt atggacagcg
     4861 cagaaagagc tggggagaat tgtgaaattg ttatccgctc acaattccac acaacatacg
     4921 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     4981 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg
     5041 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct
     5101 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc
     5161 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg
     5221 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg
     5281 cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
     5341 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac
     5401 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
     5461 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt
     5521 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc
     5581 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag
     5641 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac
     5701 tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
     5761 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
     5821 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg
     5881 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
     5941 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     6001 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     6061 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     6121 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     6181 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     6241 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     6301 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     6361 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6421 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6481 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6541 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6601 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6661 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6721 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6781 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6841 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6901 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     6961 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     7021 ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt
     7081 tcgtctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     7141 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     7201 gggtgttggc gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag
     7261 agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     7321 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     7381 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     7441 agggttttcc cagtcacgac gttgtaaaac gacggcgcaa ggaatggtgc atgcaaggag
     7501 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     7561 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     7621 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggcga
     7681 ttagtccaat ttgttaaaga caggatatca gtggtccagg ctctagtttt gactcaacaa
     7741 tatcaccagc tgaagcctat agagtacgag ccatagataa aataaaagat tttatttagt
     7801 ctccagaaaa agggggg
//
