LOCUS       sequence-482411-        8195 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Mammalian expression of human HSP70 with a N-terminal 3x-FLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8195)
  AUTHORS   Stewart M, Paththamperuma C, McCann C, Cottingim K, Zhang H, 
            DelVecchio R, Peng I, Fennimore E, Nix JC, Saeed MN, George K, 
            Makaroff M, Colie M, Paulakonis E, Almeida MF, Afolayan AJ, Brown 
            NG, Page RC, Schisler JC
  TITLE     Phosphorylation-State Modulated Binding of HSP70: Structural 
            Insights and Compensatory Protein Engineering
  JOURNAL   bioRxiv 2025.02.17.637997
REFERENCE   2  (bases 1 to 8195)
  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"; journalName: "bioRxiv 
            2025.02.17.637997"
COMMENT     Sequence Label: pHE111-3xFlag-HSP70
FEATURES             Location/Qualifiers
     source          1..8195
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     126..148
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     140..157
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     852..872
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             931..996
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    2954..3576
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3153..3172)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(3600..3620)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3618..3915
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     CDS             3969..4763
                     /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(4023..4042)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4633..4652
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5415..5549
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5452..5471)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5506..5525
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5554..5573)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5555..5573)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5587..5603)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5587..5603)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5600..5622)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5611..5627
                     /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(5635..5641,5642..5659,5660..5665))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5680..5701
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5818..5835)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5989..6577)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6069..6088)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6748..7539,7540..7608))
                     /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     7371..7390
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7609..7713)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      7740..8168
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7827..7846)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8037..8058
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa
      181 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta
      241 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      301 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
      361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      541 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      841 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      901 tcgtttagtg aaccgtcaga attaaccatg gactacaaag accatgacgg tgattataaa
      961 gatcatgaca tcgattacaa ggatgacgat gacaagcttg cggccgcgaa ttcaatggct
     1021 aaagcagcgg cgatcggcat cgacctgggc accacctact cctgcgtggg ggtgttccaa
     1081 cacggcaagg tggagatcat cgccaacgac cagggcaacc gcaccacccc cagctacgtg
     1141 gccttcacgg acaccgagcg gctcatcggg gatgcggcca agaaccaggt ggcgctgaac
     1201 ccgcagaaca ccgtgtttga cgcgaagcgg ctgatcggcc gcaagttcgg cgacccggtg
     1261 gtgcagtcgg acatgaagca ctggcctttc caggtgatca acgacggaga caagcccaag
     1321 gtgcaggtga gctacaaggg ggagaccaag gcattctacc ccgaggagat ctcgtccatg
     1381 gtgctgacca agatgaagga gatcgccgag gcgtacctgg gctacccggt gaccaacgcg
     1441 gtgatcaccg tgccggccta cttcaacgac tcgcagcgcc aggccaccaa ggatgcgggt
     1501 gtgatcgcgg ggctcaacgt gctgcggatc atcaacgagc ccacggccgc cgccatcgcc
     1561 tacggcctgg acagaacggg caagggggag cgcaacgtgc tcatctttga cctgggcggg
     1621 ggcaccttcg acgtgtccat cctgacgatc gacgacggca tcttcgaggt gaaggccacg
     1681 gccggggaca cccacctggg tggggaggac tttgacaaca ggctggtgaa ccacttcgtg
     1741 gaggagttca agagaaaaca caagaaggac atcagccaga acaagcgagc cgtgaggcgg
     1801 ctgcgcaccg cctgcgagag ggccaagagg accctgtcgt ccagcaccca ggccagcctg
     1861 gagatcgact ccctgtttga gggcatcgac ttctacacgt ccatcaccag ggcgaggttc
     1921 gaggagctgt gctccgacct gttccgaagc accctggagc ccgtggagaa ggctctgcgc
     1981 gacgccaagc tggacaaggc ccagattcac gacctggtcc tggtcggggg ctccacccgc
     2041 atccccaagg tgcagaagct gctgcaggac ttcttcaacg ggcgcgacct gaacaagagc
     2101 atcaaccccg acgaggctgt ggcctacggg gcggcggtgc aggcggccat cctgatgggg
     2161 gacaagtccg agaacgtgca ggacctgctg ctgctggacg tggctcccct gtcgctgggg
     2221 ctggagacgg ccggaggcgt gatgactgcc ctgatcaagc gcaactccac catccccacc
     2281 aagcagacgc agatcttcac cacctactcc gacaaccaac ccggggtgct gatccaggtg
     2341 tacgagggcg agagggccat gacgaaagac aacaatctgt tggggcgctt cgagctgagc
     2401 ggcatccctc cggcccccag gggcgtgccc cagatcgagg tgaccttcga catcgatgcc
     2461 aacggcatcc tgaacgtcac ggccacggac aagagcaccg gcaaggccaa caagatcacc
     2521 atcaccaacg acaagggccg cctgagcaag gaggagatcg agcgcatggt gcaggaggcg
     2581 gagaagtaca aagcggagga cgaggtgcag cgcgagaggg tgtcagccaa gaacgccctg
     2641 gagtcctacg ccttcaacat gaagagcgcc gtggaggatg aggggctcaa gggcaagatc
     2701 agcgaggccg acaagaagaa ggtgctggac aagtgtcaag aggtcatctc gtggctggac
     2761 gccaacacct tggccgagaa ggacgagttt gagcacaaga ggaaggagct ggagcaggtg
     2821 tgtaacccca tcatcagcgg actgtaccag ggtgccggtg gtcccgggcc tgggggcttc
     2881 ggggctcagg gtcccaaggg agggtctggg tcaggcccca ccattgagga ggtagattag
     2941 tctagaggat cccgggtggc atccctgtga cccctcccca gtgcctctcc tggccctgga
     3001 agttgccact ccagtgccca ccagccttgt cctaataaaa ttaagttgca tcattttgtc
     3061 tgactaggtg tccttctata atattatggg gtggaggggg gtggtatgga gcaaggggca
     3121 agttgggaag acaacctgta gggcctgcgg ggtctattgg gaaccaagct ggagtgcagt
     3181 ggcacaatct tggctcactg caatctccgc ctcctgggtt caagcgattc tcctgcctca
     3241 gcctcccgag ttgttgggat tccaggcatg catgaccagg ctcagctaat ttttgttttt
     3301 ttggtagaga cggggtttca ccatattggc caggctggtc tccaactcct aatctcaggt
     3361 gatctaccca ccttggcctc ccaaattgct gggattacag gcgtgaacca ctgctccctt
     3421 ccctgtcctt ctgattttaa aataactata ccagcaggag gacgtccaga cacagcatag
     3481 gctacctggc catgcccaac cggtgggaca tttgagttgc ttgcttggca ctgtcctctc
     3541 atgcgttggg tccactcagt agatgcctgt tgaattgggt acgcggccag cttggctgtg
     3601 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     3661 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     3721 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     3781 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     3841 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     3901 aggaggcttt tttggaggaa ttgatcagct tgggatctga tcaagagaca ggatgaggat
     3961 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     4021 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4081 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4141 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4201 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4261 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4321 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4381 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4441 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     4501 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4561 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4621 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4681 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4741 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4801 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4861 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4921 gttcttcgcc caccccgggc tcgatcccct cgcgagttgg ttcagctgct gcctgaggct
     4981 ggacgacctc gcggagttct accggcagtg caaatccgtc ggcatccagg aaaccagcag
     5041 cggctatccg cgcatccatg cccccgaact gcaggagtgg ggaggcacga tggccgcttt
     5101 ggtcgacccg gacgggacgc tcctgcgcct gatacagaac gaattgcttg caggcatctc
     5161 atgagtgtgt cttcccgttt tccgcctgag gtcactgcgt ggatggagcg ctggcgcctg
     5221 ctgcgcgacg gcgagctgct caccacccac tcgccaagct ggaaccgtaa aaaggccgcg
     5281 ttgctggcgt ttttccatag gctccgccga tcataatcag ccataccaca tttgtagagg
     5341 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg
     5401 caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5461 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5521 tcatcaatgt atcttatcat gtctggatca attccctata gtgagtcgta ttaaattcgt
     5581 aatcatgtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5641 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5701 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5761 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5821 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5881 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5941 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     6001 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     6061 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     6121 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     6181 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     6241 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     6301 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     6361 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     6421 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     6481 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     6541 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6601 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6661 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6721 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6781 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6841 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6901 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6961 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     7021 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     7081 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     7141 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     7201 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     7261 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     7321 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     7381 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     7441 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     7501 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     7561 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7621 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7681 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     7741 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     7801 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     7861 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     7921 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     7981 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     8041 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     8101 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     8161 cgcgaatttt aacaaaatat taacgcttac aattt
//
