LOCUS       sequence-104510-        5630 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expression of HSPB3 (small HSP) tagged with V5 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5630)
  AUTHORS   Vos MJ, Zijlstra MP, Kanon B, van Waarde-Verhagen MA, Brunt ER, 
            Oosterveld-Hut HM, Carra S, Sibon OC, Kampinga HH
  TITLE     HSPB7 is the most potent polyQ aggregation suppressor within the 
            HSPB family of molecular chaperones.
  JOURNAL   Hum Mol Genet. 2010 Dec 1;19(23):4677-93. doi: 10.1093/hmg/ddq398. 
            Epub 2010 Sep 15.
  PUBMED    20843828
REFERENCE   2  (bases 1 to 5630)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1093/hmg/ddq398"; journalName: "Hum Mol Genet"; date: 
            "2010-12-1- 1"; volume: "19"; issue: "23"; pages: "4677-93"
COMMENT     Sequence Label: Frt-V5-hspB3
FEATURES             Location/Qualifiers
     source          1..5630
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             996..1037
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(1582..1599)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1588..1812
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1945..1964)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2096..2143
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             2151..3173
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2151..3173
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3303..3424
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3340..3359)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3394..3413
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3473..3489)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3473..3489)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3486..3508)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3497..3513
                     /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(3521..3527,3528..3545,3546..3551))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3566..3587
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3704..3721)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3875..4463)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3955..3974)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4634..5425,5426..5494))
                     /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     5257..5276
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5495..5599)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag cttgaattca ccatgggtaa gcctatccct aaccctctcc
     1021 tcggtctcga ttctacggga tccaaaagcg gccgcaccat ggcaaaaatc attttgaggc
     1081 acctcataga gattccagtg cgttaccagg aagagtttga agctcgaggt ctagaagact
     1141 gcaggctgga tcatgcttta tatgcactgc ctgggccaac catcgtggac ctgaggaaaa
     1201 ccagggcagc gcagtctcct ccagtggact cagcggcaga gacgccaccc cgagaaggca
     1261 aatcccactt tcagatcctg ctggacgtgg tccagttcct ccctgaagac atcatcattc
     1321 agaccttcga aggctggcta ctgataaaag cacaacacgg aaccagaatg gatgagcacg
     1381 gttttatctc aagaagcttc acccgacagt acaaactacc agatggcgtg gaaatcaaag
     1441 atttgtctgc agtcctctgt catgatggaa ttttggtggt ggaagtaaag gatccagttg
     1501 ggactaagtg acatcgtatc ggttcctgtt cagaacgcgc caaaatcgag tctagagggc
     1561 ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt
     1621 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     1681 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     1741 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg
     1801 tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacg
     1861 cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta
     1921 cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt
     1981 tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg
     2041 ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt acctagaagt
     2101 tcctattccg aagttcctat tctctagaaa gtataggaac ttccttggcc aaaaagcctg
     2161 aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc
     2221 tgatgcagct ctcggagggc gaagaatctc gtgctttcag cttcgatgta ggagggcgtg
     2281 gatatgtcct gcgggtaaat agctgcgccg atggtttcta caaagatcgt tatgtttatc
     2341 ggcactttgc atcggccgcg ctcccgattc cggaagtgct tgacattggg gaattcagcg
     2401 agagcctgac ctattgcatc tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg
     2461 aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg
     2521 ccgatcttag ccagacgagc gggttcggcc cattcggacc gcaaggaatc ggtcaataca
     2581 ctacatggcg tgatttcata tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg
     2641 tgatggacga caccgtcagt gcgtccgtcg cgcaggctct cgatgagctg atgctttggg
     2701 ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc
     2761 tgacggacaa tggccgcata acagcggtca ttgactggag cgaggcgatg ttcggggatt
     2821 cccaatacga ggtcgccaac atcttcttct ggaggccgtg gttggcttgt atggagcagc
     2881 agacgcgcta cttcgagcgg aggcatccgg agcttgcagg atcgccgcgg ctccgggcgt
     2941 atatgctccg cattggtctt gaccaactct atcagagctt ggttgacggc aatttcgatg
     3001 atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg atccggagcc gggactgtcg
     3061 ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac cgatggctgt gtagaagtac
     3121 tcgccgatag tggaaaccga cgccccagca ctcgtccgag ggcaaaggaa tagcacgtac
     3181 tacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     3241 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     3301 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     3361 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     3421 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     3481 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     3541 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     3601 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     3661 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     3721 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     3781 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     3841 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     3901 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     3961 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     4021 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     4081 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     4141 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     4201 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     4261 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     4321 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     4381 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     4441 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     4501 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     4561 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     4621 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     4681 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     4741 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     4801 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     4861 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     4921 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     4981 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     5041 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     5101 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     5161 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     5221 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     5281 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     5341 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     5401 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     5461 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     5521 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     5581 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
//
