LOCUS       sequence-482343-        6850 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Expresses GRP78 Delta KDEL fused to the GST Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6850)
  AUTHORS   Tseng CC, Zhang P, Lee AS
  TITLE     The COOH-Terminal Proline-Rich Region of GRP78 Is a Key Regulator of
            Its Cell Surface Expression and Viability of Tamoxifen-Resistant 
            Breast Cancer Cells.
  JOURNAL   Neoplasia. 2019 Aug;21(8):837-848. doi: 10.1016/j.neo.2019.05.008. 
            Epub 2019 Jul 12.
  PUBMED    31306849
REFERENCE   2  (bases 1 to 6850)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 7, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.neo.2019.05.008"; journalName: "Neoplasia"; date: 
            "2019-08"; volume: "21"; issue: "8"; pages: "837-848"
COMMENT     Sequence Label: GST-GRP78-Delta KDEL (pGEX4T1)
FEATURES             Location/Qualifiers
     source          1..6850
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(181..186,187..202,203..209)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    217..233
                     /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)."
     CDS             256..909
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     867..889
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             916..933
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(2898..2920)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3065..3083)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3151..3255
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3256..3324,3325..4116)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3474..3493)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4287..4875
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4776..4795
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5029..5046
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5119..5196
                     /gene="lacI"
                     /label=lacI promoter
     CDS             5197..6279
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(5216..5235)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(6328..6333,6334..6351,6352..6358)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6366..6382
                     /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     6390..6406
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6390..6406
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6402..6850,1..79)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6422..6439)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6422..6438)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6431..6453)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cttatcgact gcacggtgca ccaatgcttc tggcgtcagg cagccatcgg aagctgtggt
       61 atggctgtgc aggtcgtaaa tcactgcata attcgtgtcg ctcaaggcgc actcccgttc
      121 tggataatgt tttttgcgcc gacatcataa cggttctggc aaatattctg aaatgagctg
      181 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
      241 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      301 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      361 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      421 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      481 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      541 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      601 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      661 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      721 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      781 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      841 agcaagtata tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat
      901 cctccaaaat cggatctggt tccgcgtgga tccgaggagg aggacaagaa ggaggacgtg
      961 ggcacggtgg tcggcatcga cctggggacc acctactcct gcgtcggcgt gttcaagaac
     1021 ggccgcgtgg agatcatcgc caacgatcag ggcaaccgca tcacgccgtc ctatgtcgcc
     1081 ttcactcctg aaggggaacg tctgattggc gatgccgcca agaaccagct cacctccaac
     1141 cccgagaaca cggtctttga cgccaagcgg ctcatcggcc gcacgtggaa tgacccgtct
     1201 gtgcagcagg acatcaagtt cttgccgttc aaggtggttg aaaagaaaac taaaccatac
     1261 attcaagttg atattggagg tgggcaaaca aagacatttg ctcctgaaga aatttctgcc
     1321 atggttctca ctaaaatgaa agaaaccgct gaggcttatt tgggaaagaa ggttacccat
     1381 gcagttgtta ctgtaccagc ctattttaat gatgcccaac gccaagcaac caaagacgct
     1441 ggaactattg ctggcctaaa tgttatgagg atcatcaacg agcctacggc agctgctatt
     1501 gcttatggcc tggataagag ggagggggag aagaacatcc tggtgtttga cctgggtggc
     1561 ggaaccttcg atgtgtctct tctcaccatt gacaatggtg tcttcgaagt tgtggccact
     1621 aatggagata ctcatctggg tggagaagac tttgaccagc gtgtcatgga acacttcatc
     1681 aaactgtaca aaaagaagac gggcaaagat gtcaggaaag acaatagagc tgtgcagaaa
     1741 ctccggcgcg aggtagaaaa ggccaaacgg gccctgtctt ctcagcatca agcaagaatt
     1801 gaaattgagt ccttctatga aggagaagac ttttctgaga ccctgactcg ggccaaattt
     1861 gaagagctca acatggatct gttccggtct actatgaagc ccgtccagaa agtgttggaa
     1921 gattctgatt tgaagaagtc tgatattgat gaaattgttc ttgttggtgg ctcgactcga
     1981 attccaaaga ttcagcaact ggttaaagag ttcttcaatg gcaaggaacc atcccgtggc
     2041 ataaacccag atgaagctgt agcgtatggt gctgctgtcc aggctggtgt gctctctggt
     2101 gatcaagata caggtgacct ggtactgctt gatgtatgtc cccttacact tggtattgaa
     2161 actgtgggag gtgtcatgac caaactgatt ccaaggaaca cagtggtgcc taccaagaag
     2221 tctcagatct tttctacagc ttctgataat caaccaactg ttacaatcaa ggtctatgaa
     2281 ggtgaaagac ccctgacaaa agacaatcat cttctgggta catttgatct gactggaatt
     2341 cctcctgctc ctcgtggggt cccacagatt gaagtcacct ttgagataga tgtgaatggt
     2401 attcttcgag tgacagctga agacaagggt acagggaaca aaaataagat cacaatcacc
     2461 aatgaccaga atcgcctgac acctgaagaa atcgaaagga tggttaatga tgctgagaag
     2521 tttgctgagg aagacaaaaa gctcaaggag cgcattgata ctagaaatga gttggaaagc
     2581 tatgcctatt ctctaaagaa tcagattgga gataaagaaa agctgggagg taaactttcc
     2641 tctgaagata aggagaccat ggaaaaagct gtagaagaaa agattgaatg gctggaaagc
     2701 caccaagatg ctgacattga agacttcaaa gctaagaaga aggaactgga agaaattgtt
     2761 caaccaatta tcagcaaact ctatggaagt gcaggccctc ccccaactgg tgaagaggat
     2821 acagcagaat agctcgagcg gccgcatcgt gactgactga cgatctgcct cgcgcgtttc
     2881 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     2941 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     3001 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtataattc ttgaagacga
     3061 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     3121 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     3181 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     3241 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     3301 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     3361 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     3421 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     3481 ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat
     3541 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     3601 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     3661 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     3721 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     3781 cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa
     3841 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     3901 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     3961 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     4021 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     4081 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     4141 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     4201 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     4261 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     4321 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     4381 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
     4441 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     4501 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     4561 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     4621 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     4681 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     4741 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     4801 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     4861 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     4921 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     4981 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     5041 agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
     5101 tcacaccgca taaattccga caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga
     5161 tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac gttatacgat
     5221 gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa ccaggccagc
     5281 cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct gaattacatt
     5341 cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg cgttgccacc
     5401 tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat
     5461 caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa
     5521 gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg
     5581 gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc gttatttctt
     5641 gatgtctctg accagacacc catcaacagt attattttct cccatgaaga cggtacgcga
     5701 ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt agcgggccca
     5761 ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct cactcgcaat
     5821 caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg ttttcaacaa
     5881 accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc caacgatcag
     5941 atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg tgcggatatc
     6001 tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc gttaaccacc
     6061 atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct gcaactctct
     6121 cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa aagaaaaacc
     6181 accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     6241 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag
     6301 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     6361 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacggatt
     6421 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     6481 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     6541 gcccttccca acagttgcgc agcctgaatg gcgaatggcg ctttgcctgg tttccggcac
     6601 cagaagcggt gccggaaagc tggctggagt gcgatcttcc tgaggccgat actgtcgtcg
     6661 tcccctcaaa ctggcagatg cacggttacg atgcgcccat ctacaccaac gtgacctatc
     6721 ccattacggt caatccgccg tttgttccca cggagaatcc gacgggttgt tactcgctca
     6781 catttaatgt tgatgaaagc tggctacagg aaggccagac gcgaattatt tttgatggcg
     6841 ttggaattag
//
