LOCUS       sequence-454082-        6611 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  expressed human Hsp70 (K71E mutation) with a GFP fluorescent protein
            tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6611)
  AUTHORS   French JB, Zhao H, An S, Niessen S, Deng Y, Cravatt BF, Benkovic SJ
  TITLE     Hsp70/Hsp90 chaperone machinery is involved in the assembly of the 
            purinosome.
  JOURNAL   Proc Natl Acad Sci U S A. 2013 Feb 12;110(7):2528-33. doi: 
            10.1073/pnas.1300173110. Epub 2013 Jan 28.
  PUBMED    23359685
REFERENCE   2  (bases 1 to 6611)
  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"; doi: 
            "10.1073/pnas.1300173110"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2013-02-12- 12"; volume: "110"; issue: "7"; pages: "2528-33"
COMMENT     Sequence Label: pHsp70(K71E)-GFP
FEATURES             Location/Qualifiers
     source          1..6611
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        61..364
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        365..568
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     519..539
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2551..2560
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2557..2559,2560..2562,2563..3276)
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLK
                     ERVTAAGITLGMDELYK"
     primer_bind     complement(2602..2623)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     polyA_signal    3399..3520
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3436..3455)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3490..3509
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3527..3982)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3664..3685)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3876..3895
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4009..4113
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4115..4472
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4115..4306
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4138..4158)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4323..4458
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4507..5301
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4507..5301
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4561..4580)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5171..5190
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5489..5508)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5533..5580
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5909..6497
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6398..6417
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
       61 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
      121 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
      181 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
      241 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
      301 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac
      361 catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
      421 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
      481 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
      541 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgatg
      601 gccaaagccg cggcgatcgg catcgacctg ggcaccacct actcctgcgt gggggtgttc
      661 caacacggca aggtggagat catcgccaac gaccagggca accgcaccac ccccagctac
      721 gtggccttca cggacaccga gcggctcatc ggggatgcgg ccaagaacca ggtggcgctg
      781 aacccgcaga acaccgtgtt tgacgcggag cggctgatcg gccgcaagtt cggcgacccg
      841 gtggtgcagt cggacatgaa gcactggcct ttccaggtga tcaacgacgg agacaagccc
      901 aaggtgcagg tgagctacaa gggggagacc aaggcattct accccgagga gatctcgtcc
      961 atggtgctga ccaagatgaa ggagatcgcc gaggcgtacc tgggctaccc ggtgaccaac
     1021 gcggtgatca ccgtgccggc ctacttcaac gactcgcagc gccaggccac caaggatgcg
     1081 ggtgtgatcg cggggctcaa cgtgctgcgg atcatcaacg agcccacggc cgccgccatc
     1141 gcctacggcc tggacagaac gggcaagggg gagcgcaacg tgctcatctt tgacctgggc
     1201 gggggcacct tcgacgtgtc catcctgacg atcgacgacg gcatcttcga ggtgaaggcc
     1261 acggccgggg acacccacct gggtggggag gactttgaca acaggctggt gaaccacttc
     1321 gtggaggagt tcaagagaaa acacaagaag gacatcagcc agaacaagcg agccgtgagg
     1381 cggctgcgca ccgcctgcga gagggccaag aggaccctgt cgtccagcac ccaggccagc
     1441 ctggagatcg actccctgtt tgagggcatc gacttctaca cgtccatcac cagggcgagg
     1501 ttcgaggagc tgtgctccga cctgttccga agcaccctgg agcccgtgga gaaggctctg
     1561 cgcgacgcca agctggacaa ggcccagatt cacgacctgg tcctggtcgg gggctccacc
     1621 cgcatcccca aggtgcagaa gctgctgcag gacttcttca acgggcgcga cctgaacaag
     1681 agcatcaacc ccgacgaggc tgtggcctac ggggcggcgg tgcaggcggc catcctgatg
     1741 ggggacaagt ccgagaacgt gcaggacctg ctgctgctgg acgtggctcc cctgtcgctg
     1801 gggctggaga cggccggagg cgtgatgact gccctgatca agcgcaactc caccatcccc
     1861 accaagcaga cgcagatctt caccacctac tccgacaacc aacccggggt gctgatccag
     1921 gtgtacgagg gcgagagggc catgacgaaa gacaacaatc tgttggggcg cttcgagctg
     1981 agcggcatcc ctccggcccc caggggcgtg ccccagatcg aggtgacctt cgacatcgat
     2041 gccaacggca tcctgaacgt cacggccacg gacaagagca ccggcaaggc caacaagatc
     2101 accatcacca acgacaaggg ccgcctgagc aaggaggaga tcgagcgcat ggtgcaggag
     2161 gcggagaagt acaaagcgga ggacgaggtg cagcgcgaga gggtgtcagc caagaacgcc
     2221 ctggagtcct acgccttcaa catgaagagc gccgtggagg atgaggggct caagggcaag
     2281 atcagcgagg cggacaagaa gaaggttctg gacaagtgtc aagaggtcat ctcgtggctg
     2341 gacgccaaca ccttggccga gaaggacgag tttgagcaca agaggaagga gctggagcag
     2401 gtgtgtaacc ccatcatcag cggactgtac cagggtgccg gtggtcccgg gcctgggggc
     2461 ttcggggctc agggtcccaa gggagggtct gggtcaggcc ccaccattga ggaggtggat
     2521 acggtaccgc gggcccggga tccaccggtc gccaccatgg tgagcaaggg cgaggagctg
     2581 ttcaccgggg tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc
     2641 agcgtgtccg gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc
     2701 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc
     2761 gtgcagtgct tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
     2821 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
     2881 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
     2941 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc
     3001 cacaacgtct atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc
     3061 cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc
     3121 atcggcgacg gtcccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg
     3181 agcaaagacc ccaacgagaa gcgcgatcac atggtcctga aggagcgcgt gaccgccgcc
     3241 gggatcactc tcggcatgga cgagctgtac aagtaaagcg gccgcgactc tagatcataa
     3301 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     3361 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     3421 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     3481 attctagttg tggtttgtcc aaactcatca atgtatctta aggcgtaaat tgtaagcgtt
     3541 aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag
     3601 gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt
     3661 gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga
     3721 aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg
     3781 gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct
     3841 tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc
     3901 gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt
     3961 aatgcgccgc tacagggcgc gtcaggtggc acttttcggg gaaatgtgcg cggaacccct
     4021 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     4081 taaatgcttc aataatattg aaaaaggaag agtcctgagg cggaaagaac cagctgtgga
     4141 atgtgtgtca gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     4201 gcatgcatct caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca
     4261 gaagtatgca aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc
     4321 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     4381 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     4441 gaggcttttt tggaggccta ggcttttgca aagatcgatc aagagacagg atgaggatcg
     4501 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     4561 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     4621 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4681 gaactgcaag acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4741 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4801 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4861 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4921 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4981 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg
     5041 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     5101 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     5161 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     5221 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     5281 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     5341 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5401 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5461 tcttcgccca ccctaggggg aggctaactg aaacacggaa ggagacaata ccggaaggaa
     5521 cccgcgctat gacggcaata aaaagacaga ataaaacgca cggtgttggg tcgtttgttc
     5581 ataaacgcgg ggttcggtcc cagggctggc actctgtcga taccccaccg agaccccatt
     5641 ggggccaata cgcccgcgtt tcttcctttt ccccacccca ccccccaagt tcgggtgaag
     5701 gcccagggct cgcagccaac gtcggggcgg caggccctgc catagcctca ggttactcat
     5761 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     5821 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     5881 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5941 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6001 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     6061 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     6121 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     6181 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     6241 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6301 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6361 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6421 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6481 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     6541 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     6601 ccgccatgca t
//
