LOCUS       sequence-429022-        6718 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of human CALR wt-Delta-KDEL-FLAG-KDEL.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6718)
  TITLE     Masubuchi et al., 2024
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6718)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pcDNA3.1/CALR wt-Delta-KDEL-FLAG-KDEL
FEATURES             Location/Qualifiers
     source          1..6718
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(320..339)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(321..339)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(384..587)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(413..433)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        588..967
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     1139..1158
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1233..1337
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1338..1406,1407..2198)
                     /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     complement(1556..1575)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2369..2957
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2858..2877
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3111..3128
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3245..3266
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3281..3286,3287..3304,3305..3311)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3319..3335
                     /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)."
     primer_bind     3324..3346
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3343..3359
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3343..3359
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    3408..3529
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3419..3438)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3473..3492
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(3703..4497)
                     /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(3814..3833)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4424..4443
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(4564..4893)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4578..4713
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4632..4651)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     4878..4898
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(4907..5335)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5017..5038)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5229..5248
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    5381..5605
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5594..5611
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(5694..5717)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
ORIGIN
        1 tggccgacag agcataaaag cgtgcatcct ggcttgtctg caaaccttta tctttctcct
       61 cgtcaccgta gaacttgccg gaactgagaa cgaatttgcc aaaatctgac ttgtgtttgg
      121 attcgatcca gcgggaagtc cacccgtctc cgtccagaaa ctgctccttg aagtagacgg
      181 caggctcggc gacggccagg ccgaggaggc cgagcagcag cggcacggat agcagcatgg
      241 tggcgaattc caccacactg gactagtgga tccgagctcg gtaccaagct taagtttaaa
      301 cgctagccag cttgggtctc cctatagtga gtcgtattaa tttcgataag ccagtaagca
      361 gtgggttctc tagttagcca gagagctctg cttatataga cctcccaccg tacacgccta
      421 ccgcccattt gcgtcaatgg ggcggagttg ttacgacatt ttggaaagtc ccgttgattt
      481 tggtgccaaa acaaactccc attgacgtca atggggtgga gacttggaaa tccccgtgag
      541 tcaaaccgct atccacgccc attgatgtac tgccaaaacc gcatcaccat ggtaatagcg
      601 atgactaata cgtagatgta ctgccaagta ggaaagtccc ataaggtcat gtactgggca
      661 taatgccagg cgggccattt accgtcattg acgtcaatag ggggcgtact tggcatatga
      721 tacacttgat gtactgccaa gtgggcagtt taccgtaaat actccaccca ttgacgtcaa
      781 tggaaagtcc ctattggcgt tactatggga acatacgtca ttattgacgt caatgggcgg
      841 gggtcgttgg gcggtcagcc aggcgggcca tttaccgtaa gttatgtaac gcggaactcc
      901 atatatgggc tatgaactaa tgaccccgta attgattact attaataact agtcaataat
      961 caatgtcaac gcgtatatct ggcccgtaca tcgcgaagca gcgcaaaacg cctaacccta
     1021 agcagattct tcatgcaatt gtcggtcaag ccttgccttg ttgtagctta aattttgctc
     1081 gcgcactact cagcgacctc caacacacaa gcagggagca gatactggct taactatgcg
     1141 gcatcagagc agattgtact gagagtgcac cataggggat cgggagatct cccgatccgt
     1201 cgacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     1261 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     1321 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     1381 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     1441 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     1501 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     1561 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     1621 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     1681 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     1741 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     1801 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     1861 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     1921 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     1981 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     2041 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     2101 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     2161 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     2221 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     2281 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     2341 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     2401 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     2461 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     2521 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     2581 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     2641 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     2701 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     2761 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     2821 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     2881 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     2941 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     3001 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     3061 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     3121 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     3181 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     3241 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     3301 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     3361 atgattacgc caagctctag ctagaggtcg acggtataca gacatgataa gatacattga
     3421 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     3481 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagttg gggtgggcga
     3541 agaactccag catgagatcc ccgcgctgga ggatcatcca gccggcgtcc cggaaaacga
     3601 ttccgaagcc caacctttca tagaaggcgg cggtggaatc gaaatctcgt gatggcaggt
     3661 tgggcgtcgc ttggtcggtc atttcgaacc ccagagtccc gctcagaaga actcgtcaag
     3721 aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa
     3781 gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca acgctatgtc
     3841 ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa agcggccatt
     3901 ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat cctcgccgtc
     3961 gggcatgcgc gccttgagcc tggcgaacag ttcggctggc gcgagcccct gatgctcttc
     4021 gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc gctcgatgcg
     4081 atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca gccgccgcat
     4141 tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca ggagatcctg
     4201 ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa cgtcgagcac
     4261 agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct cgtcctgcag
     4321 ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc cctgcgctga
     4381 cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt catagccgaa
     4441 tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt caatcatgcg
     4501 aaacgatcct catcctgtct cttgatcaga tccgaaaatg gatatacaag ctcccgggag
     4561 ctttttgcaa aagcctaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga
     4621 ggcagaggcg gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga
     4681 atgggcggaa ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggactatggt
     4741 tgctgactaa ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt
     4801 tccacacctg gttgctgact aattgagatg catgctttgc atacttctgc ctgctgggga
     4861 gcctggggac tttccacacc ctaactgaca cacattccac agaattaatt cgcgttaaat
     4921 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     4981 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     5041 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     5101 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     5161 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     5221 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     5281 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtgggga
     5341 taccccctag agccccagct ggttctttcc gcctcagaag ccatagagcc caccgcatcc
     5401 ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc caccccaccc
     5461 cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt ttattaggaa
     5521 aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg gcaaacaaca
     5581 gatggctggc aactagaagg cacagtcgag gctgatcagc gggtttaaac gggccctcta
     5641 gactcgagcg gccgccactg tgctggatat ctgcagaatt cctacagctc gtctttatcg
     5701 tcatcgtctt tgtagtcggc ctggccgggg acatcttcct cctcatcttc ctccttgtcc
     5761 tcctcatcct cctcatcctc atctttgtcc tcatcatcct ccttgtcctc tgcctcctcc
     5821 tcctctttgc gtttcttgtc ttcttcctcc tccttaagcc tctgctcctc gtcctgtttg
     5881 tccttcattt gtttctctgc tgcctttgtt acgccccacg tctcgttgcc aaactcctca
     5941 gcgtatgcct catcgttggt gatgaggaag ttgtcaaaga tggtgccaga cttgacctgc
     6001 cagaggtcca ggcccagcac gccaaagtta tcataggcat agatactggg atcgggagaa
     6061 tactcggggt tgtcaatttc tgggtggatc caagtgccct tgtaatctgg gttgtcgatc
     6121 tgccggggct tccactcacc cttgtactca gggttctgaa tcactggggg ttcccactct
     6181 ccgtccatct cttcatccca gtcctcgggc ttcttagcat cagggtcagg gatatgctcg
     6241 ggcttgtccc agtcctcagg cttggagtct gtgggatcat cgatcttggc ccgctcatcc
     6301 cagtcttccg gttttgaagc atcaggatcc tttatcttct tgggtggcag gaagtcccaa
     6361 tcgtcttcca aggagccgga ctccacctgg ctgttgtcaa tcttcacctc ataggtgttg
     6421 tctggccgca caatcagtgt gtacaggtgt gtaaactcat catccttgca acggatgtcc
     6481 ttgttgatca gcacgttctt gcccttgtag ttgaagatga catgaacctt cttggtgcca
     6541 gggccacaga tgtcgggacc aaacatgatg ttgtattctg agtctccgtg catgtctgtc
     6601 tggtccaaac tattaggaaa cagcttcaca tagccgcccc cacagtcgat gttctgctca
     6661 tgtttcaccg tgaactgcac caccagcgtc tggcctttgt tgctgaaagg ctcgaaac
//
