LOCUS       sequence-407200-        5837 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human CALR ins5 (del1-308)-FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5837)
  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 5837)
  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: pcDNA3.1/CALR ins5 (del1-308)-FLAG
FEATURES             Location/Qualifiers
     source          1..5837
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      958..967
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1333..1356
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1428..1445)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1434..1658
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1704..2132
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1791..1810)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2001..2022
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2141..2161)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2146..2475
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2326..2461
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2388..2407
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2542..3336
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2596..2615)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3206..3225
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3510..3631
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3547..3566)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3601..3620
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3680..3696)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3680..3696)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3693..3715)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3704..3720
                     /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(3728..3734,3735..3752,3753..3758))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3773..3794
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3911..3928)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4082..4670)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4162..4181)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4841..5632,5633..5701))
                     /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     5464..5483
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5702..5806)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattcgcc
      961 accatggata actttggcgt gctgggcctg gacctctggc aggtcaagtc tggcaccatc
     1021 tttgacaact tcctcatcac caacgatgag gcatacgctg aggagtttgg caacgagacg
     1081 tggggcgtaa caaaggcagc agagaaacaa atgaaggaca aacaggacga ggagcagagg
     1141 cttaaggagg aggaagaaga caagaaacgc aaagaggagg aggaggcaga ggacaattgt
     1201 cggaggatga tgaggacaaa gatgaggatg aggaggatga ggaggacaag gaggaagatg
     1261 aggaggaaga tgtccccggc caggccaagg acgagctgta gagaggcctg cctccagggc
     1321 tggactgagg ccgactacaa ggacgatgat gacaagtgaa ttctgcagat atccagcaca
     1381 gtggcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     1441 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     1501 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     1561 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     1621 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     1681 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1741 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1801 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1861 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1921 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1981 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2041 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2101 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     2161 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     2221 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     2281 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     2341 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     2401 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     2461 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     2521 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     2581 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     2641 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     2701 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     2761 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     2821 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     2881 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     2941 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3001 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3061 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3121 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     3181 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     3241 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     3301 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     3361 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     3421 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     3481 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     3541 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3601 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     3661 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     3721 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3781 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3841 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3901 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3961 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4021 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4081 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4141 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4201 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4261 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4321 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4381 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4441 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4501 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4561 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4621 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4681 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4741 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4801 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4861 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4921 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4981 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5041 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5101 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5161 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5221 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5281 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5341 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5401 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5461 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5521 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5581 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5641 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5701 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5761 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5821 aagtgccacc tgacgtc
//
