LOCUS       sequence-476077-        7018 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GRP78 lacking the ER signal peptide in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7018)
  AUTHORS   Liu Z, Ha DP, Lin LL, Qi L, Lee AS
  TITLE     Requirements for nuclear GRP78 transcriptional regulatory activities
            and interaction with nuclear GRP94.
  JOURNAL   J Biol Chem. 2025 Feb 28;301(4):108369. doi: 
            10.1016/j.jbc.2025.108369.
  PUBMED    40024475
REFERENCE   2  (bases 1 to 7018)
  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: "J Biol 
            Chem."; date: "2025-02-28"; volume: "301(4):108369. doi"; pages: " 
            10.1016/j.jbc.2025.108369"
COMMENT     Sequence Label: FLAG-GRP78-DeltaER
FEATURES             Location/Qualifiers
     source          1..7018
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        281..484
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     481..505
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          618..1093
                     /label=beta-globin intron
                     /note="internally truncated intron from human beta-globin"
     protein_bind    1142..1166
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1167..1176
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1176..1199
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    complement(3111..3135)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    3164..3285
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3175..3194)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3229..3248
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     enhancer        3383..3762
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3763..3966
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     CDS             3999..4598
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3999..4018)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4495..4515
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4636..4653)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4642..4866
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4891..4908)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5062..5650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5142..5161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5821..6612,6613..6681))
                     /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     6444..6463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6682..6786)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6854..6872
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     protein_bind    6898..6918
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        join(6919..7018,1..280)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc
       61 catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac
      121 tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa
      181 tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac
      241 ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
      301 catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga
      361 cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa
      421 ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag
      481 agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca
      541 tagaagacac cgggaccgat ccagcctccc ctcgaagctt acatgtggta ccgagctcgg
      601 atcctgagaa cttcagggtg agtctatggg acccttgatg ttttctttcc ccttcttttc
      661 tatggttaag ttcatgtcat aggaagggga gaagtaacag ggtacacata ttgaccaaat
      721 cagggtaatt ttgcatttgt aattttaaaa aatgctttct tcttttaata tacttttttg
      781 tttatcttat ttctaatact ttccctaatc tctttctttc agggcaataa tgatacaatg
      841 tatcatgcct ctttgcacca ttctaaagaa taacagtgat aatttctggg ttaaggcaat
      901 agcaatattt ctgcatataa atatttctgc atataaattg taactgatgt aagaggtttc
      961 atattgctaa tagcagctac aatccagcta ccattctgct tttattttat ggttgggata
     1021 aggctggatt attctgagtc caagctaggc ccttttgcta atcatgttca tacctcttat
     1081 cttcctccca cagctcctgg gcaacgtgct ggtctgtgtg ctggcccatc actttggcaa
     1141 agcaagtttg tacaaaaaag caggctgcca ccatggacta caaagatgac gatgacaagg
     1201 aggaggagga caagaaggag gacgtgggca cggtggtcgg catcgacctg gggaccacct
     1261 actcctgcgt cggcgtgttc aagaacggcc gcgtggagat catcgccaac gatcagggca
     1321 accgcatcac gccgtcctat gtcgccttca ctcctgaagg ggaacgtctg attggcgatg
     1381 ccgccaagaa ccagctcacc tccaaccccg agaacacggt ctttgacgcc aagcggctca
     1441 tcggccgcac gtggaatgac ccgtctgtgc agcaggacat caagttcttg ccgttcaagg
     1501 tggttgaaaa gaaaactaaa ccatacattc aagttgatat tggaggtggg caaacaaaga
     1561 catttgctcc tgaagaaatt tctgccatgg ttctcactaa aatgaaagaa accgctgagg
     1621 cttatttggg aaagaaggtt acccatgcag ttgttactgt accagcctat tttaatgatg
     1681 cccaacgcca agcaaccaaa gacgctggaa ctattgctgg cctaaatgtt atgaggatca
     1741 tcaacgagcc tacggcagct gctattgctt atggcctgga taagagggag ggggagaaga
     1801 acatcctggt gtttgacctg ggtggcggaa ccttcgatgt gtctcttctc accattgaca
     1861 atggtgtctt cgaagttgtg gccactaatg gagatactca tctgggtgga gaagactttg
     1921 accagcgtgt catggaacac ttcatcaaac tgtacaaaaa gaagacgggc aaagatgtca
     1981 ggaaagacaa tagagctgtg cagaaactcc ggcgcgaggt agaaaaggcc aaacgggccc
     2041 tgtcttctca gcatcaagca agaattgaaa ttgagtcctt ctatgaagga gaagactttt
     2101 ctgagaccct gactcgggcc aaatttgaag agctcaacat ggatctgttc cggtctacta
     2161 tgaagcccgt ccagaaagtg ttggaagatt ctgatttgaa gaagtctgat attgatgaaa
     2221 ttgttcttgt tggtggctcg actcgaattc caaagattca gcaactggtt aaagagttct
     2281 tcaatggcaa ggaaccatcc cgtggcataa acccagatga agctgtagcg tatggtgctg
     2341 ctgtccaggc tggtgtgctc tctggtgatc aagatacagg tgacctggta ctgcttgatg
     2401 tatgtcccct tacacttggt attgaaactg tgggaggtgt catgaccaaa ctgattccaa
     2461 ggaacacagt ggtgcctacc aagaagtctc agatcttttc tacagcttct gataatcaac
     2521 caactgttac aatcaaggtc tatgaaggtg aaagacccct gacaaaagac aatcatcttc
     2581 tgggtacatt tgatctgact ggaattcctc ctgctcctcg tggggtccca cagattgaag
     2641 tcacctttga gatagatgtg aatggtattc ttcgagtgac agctgaagac aagggtacag
     2701 ggaacaaaaa taagatcaca atcaccaatg accagaatcg cctgacacct gaagaaatcg
     2761 aaaggatggt taatgatgct gagaagtttg ctgaggaaga caaaaagctc aaggagcgca
     2821 ttgatactag aaatgagttg gaaagctatg cctattctct aaagaatcag attggagata
     2881 aagaaaagct gggaggtaaa ctttcctctg aagataagga gaccatggaa aaagctgtag
     2941 aagaaaagat tgaatggctg gaaagccacc aagatgctga cattgaagac ttcaaagcta
     3001 agaagaagga actggaagaa attgttcaac caattatcag caaactctat ggaagtgcag
     3061 gccctccccc aactggtgaa gaggatacag cagaaaaaga tgagttgtag acccagcttt
     3121 cttgtacaaa gtggtgatgg ccggccgctt cgagcagaca tgataagata cattgatgag
     3181 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     3241 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     3301 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     3361 ctctacaaat gtggtacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     3421 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     3481 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     3541 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     3601 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     3661 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     3721 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
     3781 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
     3841 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac
     3901 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc
     3961 agagctctct ggctaactag agaacccact gcgccaccat gaccgagtac aagcccacgg
     4021 tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg
     4081 ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg
     4141 agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg
     4201 acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt
     4261 tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac
     4321 agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg
     4381 tcggcgtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag
     4441 tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc
     4501 tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac
     4561 cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgact cgagtctaga gggcccgttt
     4621 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     4681 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     4741 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     4801 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     4861 ctatgggcgg ccgcggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4921 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4981 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     5041 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5101 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5161 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5221 cgcctttctc tcttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5281 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5341 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5401 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5461 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5521 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5581 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5641 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5701 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5761 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5821 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5881 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5941 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6001 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6061 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6121 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6181 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6241 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6301 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6361 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6421 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6481 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6541 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6601 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6661 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6721 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6781 tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
     6841 attaacctat aaaaataggc gtatcacgag gccctttcgt cggcgcgccg cggccgccaa
     6901 ctttgtatag aaaagttgga cattgattat tgactagtta ttaatagtaa tcaattacgg
     6961 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatgg
//
