LOCUS       sequence-407202-        6394 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human CALR ins5 (del309-430)-FLAG.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6394)
  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 6394)
  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 (del309-430)-FLAG
FEATURES             Location/Qualifiers
     source          1..6394
                     /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"
     CDS             1888..1911
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1985..2002)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1991..2215
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2261..2689
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2348..2367)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2558..2579
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2698..2718)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2703..3032
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2883..3018
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2945..2964
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3099..3893
                     /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(3153..3172)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3763..3782
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4067..4188
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4104..4123)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4158..4177
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4237..4253)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4237..4253)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4250..4272)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4261..4277
                     /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(4285..4291,4292..4309,4310..4315))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4330..4351
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4468..4485)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4639..5227)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4719..4738)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5398..6189,6190..6258))
                     /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     6021..6040
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6259..6363)
                     /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 accatgctgc tatccgtgcc gctgctgctc ggcctcctcg gcctggccgt cgccgagcct
     1021 gccgtctact tcaaggagca gtttctggac ggagacgggt ggacttcccg ctggatcgaa
     1081 tccaaacaca agtcagattt tggcaaattc gttctcagtt ccggcaagtt ctacggtgac
     1141 gaggagaaag ataaaggttt gcagacaagc caggatgcac gcttttatgc tctgtcggcc
     1201 agtttcgagc ctttcagcaa caaaggccag acgctggtgg tgcagttcac ggtgaaacat
     1261 gagcagaaca tcgactgtgg gggcggctat gtgaagctgt ttcctaatag tttggaccag
     1321 acagacatgc acggagactc agaatacaac atcatgtttg gtcccgacat ctgtggccct
     1381 ggcaccaaga aggttcatgt catcttcaac tacaagggca agaacgtgct gatcaacaag
     1441 gacatccgtt gcaaggatga tgagtttaca cacctgtaca cactgattgt gcggccagac
     1501 aacacctatg aggtgaagat tgacaacagc caggtggagt ccggctcctt ggaagacgat
     1561 tgggacttcc tgccacccaa gaagataaag gatcctgatg cttcaaaacc ggaagactgg
     1621 gatgagcggg ccaagatcga tgatcccaca gactccaagc ctgaggactg ggacaagccc
     1681 gagcatatcc ctgaccctga tgctaagaag cccgaggact gggatgaaga gatggacgga
     1741 gagtgggaac ccccagtgat tcagaaccct gagtacaagg gtgagtggaa gccccggcag
     1801 atcgacaacc cagattacaa gggcacttgg atccacccag aaattgacaa ccccgagtat
     1861 tctcccgatc ccagtatcta tgcctatgac tacaaagacg atgacgataa ataggaattc
     1921 tgcagatatc cagcacagtg gcggccgctc gagtctagag ggcccgttta aacccgctga
     1981 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     2041 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     2101 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     2161 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct
     2221 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2281 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2341 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2401 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2461 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     2521 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     2581 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     2641 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga
     2701 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2761 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     2821 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     2881 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     2941 tttttattta tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag
     3001 gaggcttttt tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt
     3061 tcggatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
     3121 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
     3181 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
     3241 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
     3301 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
     3361 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
     3421 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
     3481 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
     3541 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
     3601 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     3661 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     3721 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     3781 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     3841 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     3901 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     3961 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4021 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4081 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4141 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4201 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4261 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4321 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4381 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4441 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4501 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4561 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4621 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4681 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4741 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4801 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     4861 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4921 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4981 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5041 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5101 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5161 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5221 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5281 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5341 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5401 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5461 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5521 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5581 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5641 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5701 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5761 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5821 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5881 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5941 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6001 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6061 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6121 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6181 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6241 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6301 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6361 gcgcacattt ccccgaaaag tgccacctga cgtc
//
