LOCUS       sequence-482402-        6337 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Mammalian expression of human CHIP with the synthetic K30A 
            substitution and C-terminal myc-His tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6337)
  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 6337)
  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: pAD000-CHIP-K30A-myc-His
FEATURES             Location/Qualifiers
     source          1..6337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             931..960
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             976..993
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1016..1033)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1022..1246
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1292..1720
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1379..1398)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1589..1610
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1729..1749)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1734..2063
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1914..2049
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     1976..1995
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2130..2924
                     /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(2184..2203)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2794..2813
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3100..3221
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3137..3156)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3191..3210
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3270..3286)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3270..3286)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3283..3305)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3294..3310
                     /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(3318..3324,3325..3342,3343..3348))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3363..3384
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3501..3518)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3672..4260)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3752..3771)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4431..5222,5223..5291))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EP*LNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5054..5073
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5292..5396)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5471..5490)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5662..6041
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6042..6245
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6196..6216
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6290..6309
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6290..6308
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 atgaagggca aggaggagaa ggagggcggc gcacggctgg gcgctggcgg cggaagcccc
       61 gagaagagcc cgagcgcgca ggagctcgcg gagcagggca atcgtctgtt cgtgggccga
      121 aagtacccgg aggcggcggc ctgctacggc cgcgcgatca cccggaaccc gctggtggcc
      181 gtgtattaca ccaaccgggc cttgtgctac ctgaagatgc agcagcacga gcaggccctg
      241 gccgactgcc ggcgcgccct ggagctggac gggcagtctg tgaaggcgca cttcttcctg
      301 gggcagtgcc agctggagat ggagagctat gatgaggcca tcgccaatct gcagcgagct
      361 tacagcctgg ccaaggagca gcggctgaac ttcggggacg acatccccag cgctcttcga
      421 atcgcgaaga agaagcgctg gaacagcatt gaggagcggc gcatccacca ggagagcgag
      481 ctgcactcct acctctccag gctcattgcc gcggagcgtg agagggagct ggaagagtgc
      541 cagcgaaacc acgagggtga tgaggacgac agccacgtcc gggcccagca ggcctgcatt
      601 gaggccaagc acgacaagta catggcggac atggacgagc ttttttctca ggtggatgag
      661 aagaggaaga agcgagacat ccccgactac ctgtgtggca agatcagctt tgagctgatg
      721 cgggagccgt gcatcacgcc cagtggcatc acctacgacc gcaaggacat cgaggagcac
      781 ctgcagcgtg tgggtcattt tgaccccgtg acccggagcc ccctgaccca ggaacagctc
      841 atccccaact tggctatgaa ggaggttatt gacgcattca tctctgagaa tggctgggtg
      901 gaggactact atctagaggg cccgcggttc gaacaaaaac tcatctcaga agaggatctg
      961 aatatgcata ccggtcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg
     1021 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1081 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     1141 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     1201 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     1261 agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg
     1321 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     1381 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     1441 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     1501 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     1561 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     1621 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     1681 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc
     1741 agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc
     1801 tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc
     1861 aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc
     1921 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     1981 atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga ggaggctttt
     2041 ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt ttcggatctg
     2101 atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg cacgcaggtt
     2161 ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct
     2221 gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga
     2281 ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg
     2341 ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact
     2401 ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg
     2461 agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct
     2521 gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg
     2581 gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt
     2641 tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg
     2701 cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc
     2761 ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag
     2821 agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt
     2881 cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgagcggga ctctggggtt
     2941 cgcgaaatga ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc
     3001 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     3061 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     3121 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3181 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     3241 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3301 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3361 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3421 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3481 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3541 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3601 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3661 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3721 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3781 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3841 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     3901 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     3961 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4021 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4081 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4141 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4201 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4261 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4321 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4381 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4441 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4501 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4561 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4621 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4681 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4741 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctacggt
     4801 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4861 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     4921 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     4981 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5041 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5101 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5161 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5221 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5281 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5341 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5401 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     5461 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     5521 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     5581 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     5641 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     5701 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     5761 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     5821 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     5881 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     5941 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     6001 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     6061 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     6121 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     6181 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     6241 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     6301 actataggga gacccaagct ggctagttaa gcttgcc
//
