LOCUS       sequence-482404-        6337 bp    DNA     circular SYN 07-AUG-2025
DEFINITION  Mammalian expression of human CHIP with a 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: pAD100-CHIP-myc-His
FEATURES             Location/Qualifiers
     source          1..6337
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(31..619)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(111..130)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(790..1581,1582..1650))
                     /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     1413..1432
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1651..1755)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1830..1849)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        2021..2400
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2401..2604
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2555..2575
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2649..2668
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2649..2667
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3627..3656
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3672..3689
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3712..3729)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3718..3942
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3988..4416
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4075..4094)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4285..4306
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4425..4445)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4430..4759
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4610..4745
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4672..4691
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4826..5620
                     /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(4880..4899)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5490..5509
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5796..5917
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5833..5852)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5887..5906
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5966..5982)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5966..5982)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5979..6001)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5990..6006
                     /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(6014..6020,6021..6038,6039..6044))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6059..6080
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6197..6214)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
       61 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
      121 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
      181 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
      241 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
      301 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
      361 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
      421 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
      481 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
      541 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
      601 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
      661 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
      721 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
      781 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
      841 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
      901 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
      961 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     1021 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     1081 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     1141 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     1201 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     1261 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     1321 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     1381 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     1441 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     1501 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     1561 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     1621 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     1681 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     1741 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     1801 tctcccgatc ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     1861 cagtatctgc tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa
     1921 gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt
     1981 tttgcgctgc ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt
     2041 tattaatagt aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt
     2101 acataactta cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg
     2161 tcaataatga cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg
     2221 gtggagtatt tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt
     2281 acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg
     2341 accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
     2401 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
     2461 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
     2521 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
     2581 tgggaggtct atataagcag agctctctgg ctaactagag aacccactgc ttactggctt
     2641 atcgaaatta atacgactca ctatagggag acccaagctg gctagttaag cttgccatga
     2701 agggcaagga ggagaaggag ggcggcgcac ggctgggcgc tggcggcgga agccccgaga
     2761 agagcccgag cgcgcaggag ctcaaggagc agggcaatcg tctgttcgtg ggccgaaagt
     2821 acccggaggc ggcggcctgc tacggccgcg cgatcacccg gaacccgctg gtggccgtgt
     2881 attacaccaa ccgggccttg tgctacctga agatgcagca gcacgagcag gccctggccg
     2941 actgccggcg cgccctggag ctggacgggc agtctgtgaa ggcgcacttc ttcctggggc
     3001 agtgccagct ggagatggag agctatgatg aggccatcgc caatctgcag cgagcttaca
     3061 gcctggccaa ggagcagcgg ctgaacttcg gggacgacat ccccagcgct cttcgaatcg
     3121 cgaagaagaa gcgctggaac agcattgagg agcggcgcat ccaccaggag agcgagctgc
     3181 actcctacct ctccaggctc attgccgcgg agcgtgagag ggagctggaa gagtgccagc
     3241 gaaaccacga gggtgatgag gacgacagcc acgtccgggc ccagcaggcc tgcattgagg
     3301 ccaagcacga caagtacatg gcggacatgg acgagctttt ttctcaggtg gatgagaaga
     3361 ggaagaagcg agacatcccc gactacctgt gtggcaagat cagctttgag ctgatgcggg
     3421 agccgtgcat cacgcccagt ggcatcacct acgaccgcaa ggacatcgag gagcacctgc
     3481 agcgtgtggg tcattttgac cccgtgaccc ggagccccct gacccaggaa cagctcatcc
     3541 ccaacttggc tatgaaggag gttattgacg cattcatctc tgagaatggc tgggtggagg
     3601 actactatct agagggcccg cggttcgaac aaaaactcat ctcagaagag gatctgaata
     3661 tgcataccgg tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg
     3721 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     3781 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     3841 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     3901 aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa
     3961 ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     4021 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     4081 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4141 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     4201 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     4261 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     4321 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     4381 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     4441 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     4501 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     4561 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     4621 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4681 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     4741 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     4801 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4861 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4921 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4981 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     5041 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     5101 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     5161 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     5221 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     5281 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     5341 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     5401 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     5461 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     5521 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     5581 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgcg
     5641 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     5701 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5761 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5821 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5881 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5941 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     6001 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     6061 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     6121 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     6181 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     6241 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     6301 gaaagaacat gtgagcaaaa ggccagcaaa aggccag
//
