LOCUS       sequence-472959-        7376 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GRP78 with mutated NLS in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7376)
  AUTHORS   Liu Z, Liu G, Ha DP, Wang J, Xiong M, Lee AS
  TITLE     ER chaperone GRP78/BiP translocates to the nucleus under stress and 
            acts as a transcriptional regulator.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Aug;120(31):e2303448120. doi: 
            10.1073/pnas.2303448120. Epub 2023 Jul 24.
  PUBMED    37487081
REFERENCE   2  (bases 1 to 7376)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-08"; volume: "120(31):e2303448120. 
            doi"; pages: " 10.1073/pnas.2303448120. Epub 2023 Jul 24"
COMMENT     Sequence Label: FLAG-GRP78-NLS-Mut
FEATURES             Location/Qualifiers
     source          1..7376
                     /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             967..990
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     promoter        complement(2929..2947)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(2930..2947)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(2967..2984)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2973..3197
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3243..3671
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3330..3349)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3540..3561
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3680..3700)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3685..4014
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3865..4000
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3927..3946
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4081..4875
                     /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(4135..4154)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4745..4764
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5049..5170
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5086..5105)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5140..5159
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5219..5235)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5219..5235)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5232..5254)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5243..5259
                     /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(5267..5273,5274..5291,5292..5297))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5312..5333
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5450..5467)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5621..6209)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5701..5720)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6380..7171,7172..7240))
                     /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     7003..7022
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7241..7345)
                     /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 gcttggtacc
      901 gagctcggat ccatgaagct ctccctggtg gccgcgatgc tgctgctgct cagcgcggcg
      961 cgggccgact acaaagatga cgatgacaag gaggaggagg acaagaagga ggacgtgggc
     1021 acggtggtcg gcatcgacct ggggaccacc tactcctgcg tcggcgtgtt caagaacggc
     1081 cgcgtggaga tcatcgccaa cgatcagggc aaccgcatca cgccgtccta tgtcgccttc
     1141 actcctgaag gggaacgtct gattggcgat gccgccaaga accagctcac ctccaacccc
     1201 gagaacacgg tctttgacgc caagcggctc atcggccgca cgtggaatga cccgtctgtg
     1261 cagcaggaca tcaagttctt gccgttcaag gtggttgaaa agaaaactaa accatacatt
     1321 caagttgata ttggaggtgg gcaaacaaag acatttgctc ctgaagaaat ttctgccatg
     1381 gttctcacta aaatgaaaga aaccgctgag gcttatttgg gaaagaaggt tacccatgca
     1441 gttgttactg taccagccta ttttaatgat gcccaacgcc aagcaaccaa agacgctgga
     1501 actattgctg gcctaaatgt tatgaggatc atcaacgagc ctacggcagc tgctattgct
     1561 tatggcctgg ataagaggga gggggagaag aacatcctgg tgtttgacct gggtggcgga
     1621 accttcgatg tgtctcttct caccattgac aatggtgtct tcgaagttgt ggccactaat
     1681 ggagatactc atctgggtgg agaagacttt gaccagcgtg tcatggaaca cttcatcaaa
     1741 ctgtacaaaa agaagacggg cgcagatgtc agggcagaca atagagctgt gcaggcactc
     1801 cggcgcgagg tagaaaaggc caaacgggcc ctgtcttctc agcatcaagc aagaattgaa
     1861 attgagtcct tctatgaagg agaagacttt tctgagaccc tgactcgggc caaatttgaa
     1921 gagctcaaca tggatctgtt ccggtctact atgaagcccg tccagaaagt gttggaagat
     1981 tctgatttga agaagtctga tattgatgaa attgttcttg ttggtggctc gactcgaatt
     2041 ccaaagattc agcaactggt taaagagttc ttcaatggca aggaaccatc ccgtggcata
     2101 aacccagatg aagctgtagc gtatggtgct gctgtccagg ctggtgtgct ctctggtgat
     2161 caagatacag gtgacctggt actgcttgat gtatgtcccc ttacacttgg tattgaaact
     2221 gtgggaggtg tcatgaccaa actgattcca aggaacacag tggtgcctac caagaagtct
     2281 cagatctttt ctacagcttc tgataatcaa ccaactgtta caatcaaggt ctatgaaggt
     2341 gaaagacccc tgacaaaaga caatcatctt ctgggtacat ttgatctgac tggaattcct
     2401 cctgctcctc gtggggtccc acagattgaa gtcacctttg agatagatgt gaatggtatt
     2461 cttcgagtga cagctgaaga caagggtaca gggaacaaaa ataagatcac aatcaccaat
     2521 gaccagaatc gcctgacacc tgaagaaatc gaaaggatgg ttaatgatgc tgagaagttt
     2581 gctgaggaag acaaaaagct caaggagcgc attgatacta gaaatgagtt ggaaagctat
     2641 gcctattctc taaagaatca gattggagat aaagaaaagc tgggaggtaa actttcctct
     2701 gaagataagg agaccatgga aaaagctgta gaagaaaaga ttgaatggct ggaaagccac
     2761 caagatgctg acattgaaga cttcaaagct aagaagaagg aactggaaga aattgttcaa
     2821 ccaattatca gcaaactcta tggaagtgca ggccctcccc caactggtga agaggataca
     2881 gcagaaaaag atgagttgta gctcgagcat gcatctagag ggccctattc tatagtgtca
     2941 cctaaatgct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     3001 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     3061 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     3121 tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga
     3181 tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc
     3241 ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     3301 cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     3361 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     3421 tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg
     3481 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     3541 tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt
     3601 ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt
     3661 taacgcgaat taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc
     3721 ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag
     3781 tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc
     3841 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3901 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct
     3961 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc
     4021 ccgggagctt gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc
     4081 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
     4141 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     4201 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     4261 caggacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     4321 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     4381 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     4441 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     4501 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     4561 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac
     4621 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     4681 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     4741 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     4801 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     4861 gacgagttct tctgagcggg actctggggt tcgaaatgac cgaccaagcg acgcccaacc
     4921 tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     4981 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5041 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5101 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5161 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     5221 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5281 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5341 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5401 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5461 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5521 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5581 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5641 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5701 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5761 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5821 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5881 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5941 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6001 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6061 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6121 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6181 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6241 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6301 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6361 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6421 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6481 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6541 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6601 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6661 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6721 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6781 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6841 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6901 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6961 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7021 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7081 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7141 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7201 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7261 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7321 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
