LOCUS       sequence-431789-        7601 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High expression of the 
            Clusterin(Y125F,G132E,L135K,R138E,Q139A,E142S) mutant for 
            purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7601)
  TITLE     Hartl Lab plasmids
REFERENCE   2  (bases 1 to 7601)
  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"
COMMENT     Sequence Label: pB-T-PAF-Clu DD2
FEATURES             Location/Qualifiers
     source          1..7601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(28..456)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(138..159)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     583..605
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     597..614
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     598..614
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     621..640
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        621..639
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   706..768
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1140..1158
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1177..1195
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1214..1232
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1251..1269
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1288..1306
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1325..1343
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1362..1380
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1399..1417
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1439..1463
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     complement(3009..3026)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3015..3239
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3523..3570
                     /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)."
     rep_origin      3664..3798
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3803..4405
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MGTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     4302..4322
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4639..4760
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4676..4695)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5228..5262
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(5411..5429)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5450..5466)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5450..5466)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5463..5485)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5474..5490
                     /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(5498..5504,5505..5522,5523..5528))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5543..5564
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5681..5698)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5852..6440)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5932..5951)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6611..7402,7403..7471))
                     /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     7234..7253
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7472..7576)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat
       61 tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa tagaccgaga
      121 tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca
      181 acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct
      241 aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc
      301 cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag
      361 cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca
      421 cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc aggctgcgca
      481 actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
      541 gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta
      601 aaacgacggc cagtgaattg taatacgact cactataggg cgaattggag ctcggtattc
      661 acgacagcag gctgaataat aaaaaaatta gaaactatta tttaacccta gaaagataat
      721 catattgtga cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt
      781 ctgtatatcg aggtttattt attaatttga atagatatta agttttatta tatttacact
      841 tacatactaa taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa
      901 aacaaaaact caaaatttct tctataaagt aacaaaactt ttaaacattc tctcttttac
      961 aaaaataaac ttattttgta ctttaaaaac agtcatgttg tattataaaa taagtaatta
     1021 gcttaaccta tacataatag aaacaaatta tacttattag tcagtcagaa acaactttgg
     1081 cacatatcaa tattatgctc tcgttaatta actcgaggga tccaagctgg ccgcacgtct
     1141 ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat agagaaggta
     1201 cgtctagaac gtctccctat cagtgataga gaagtcgaca cgtctcgagc tccctatcag
     1261 tgatagagaa ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc gacacgtctc
     1321 gagctcccta tcagtgatag agaaggtacg tctagaacgt ctccctatca gtgatagaga
     1381 agtcgacacg tctcgagctc cctatcagtg atagagaagg taccccctat ataagcagag
     1441 ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt gacctccata
     1501 gaagacaccg ggaccgatcc agcctccgcg gtggcggcca tcacaagttt gtacagctag
     1561 ccaccatgat gaagactctg ctgctgtttg tggggctgct gctgacctgg gagagtgggc
     1621 aggtcctggg ggaccagacg gtctcagaca atgagctcca ggaaatgtcc aatcagggaa
     1681 gtaagtacgt caataaggaa attcaaaatg ctgtcaacgg ggtgaaacag ataaagactc
     1741 tcatagaaaa aacaaacgaa gagcgcaaga cactgctcag caacctagaa gaagccaaga
     1801 agaagaaaga ggatgcccta aatgagacca gggaatcaga gacaaagctg aaggagctcc
     1861 caggagtgtg caatgagacc atgatggccc tctgggaaga gtgtaagccc tgcctgaaac
     1921 agacctgcat gaagttcttc gcacgcgtct gcagaagtga gtcaggcaag gttggcgagg
     1981 cgcttgagtc gttcctgaac cagagctcgc ccttctactt ctggatgaat ggtgaccgca
     2041 tcgactccct gctggagaac gaccggcagc agacgcacat gctggatgtc atgcaggacc
     2101 acttcagccg cgcgtccagc atcatagacg agctcttcca ggacaggttc ttcacccggg
     2161 agccccagga tacctaccac tacctgccct tcagcctgcc ccaccggagg cctcacttct
     2221 tctttcccaa gtcccgcatc gtccgcagct tgatgccctt ctctccgtac gagcccctga
     2281 acttccacgc catgttccag cccttccttg agatgataca cgaggctcag caggccatgg
     2341 acatccactt ccatagcccg gccttccagc acccgccaac agaattcata cgagaaggcg
     2401 acgatgaccg gactgtgtgc cgggagatcc gccacaactc cacgggctgc ctgcggatga
     2461 aggaccagtg tgacaagtgc cgggagatct tgtctgtgga ctgttccacc aacaacccct
     2521 cccaggctaa gctgcggcgg gagctcgacg aatccctcca ggtcgctgag aggttgacca
     2581 ggaaatacaa cgagctgcta aagtcctacc agtggaagat gctcaacacc tcctccttgc
     2641 tggagcagct gaacgagcag tttaactggg tgtcccggct ggcaaacctc acgcaaggcg
     2701 aagaccagta ctatctgcgg gtcaccacgg tggcttccca cacttctgac tcggacgttc
     2761 cttccggtgt cactgaggtg gtcgtgaagc tctttgactc tgatcccatc actgtgacgg
     2821 tccctgtaga agtctccagg aagaacccta aatttatgga gaccgtggcg gagaaagcgc
     2881 tgcaggaata ccgcaaaaag caccgggagg agtgagcggc cgcatagata actgatccag
     2941 tgtgctgggc tacgatgcat gggcccccta ggtcgagtct agagggcccg tttaaacccg
     3001 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     3061 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     3121 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     3181 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc
     3241 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg
     3301 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3361 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3421 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3481 cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag
     3541 ttcctattct ctagaaagta taggaacttc cttggccaaa aagaccggta ccaggcaggc
     3601 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     3661 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     3721 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     3781 ggaggctttt ttggaggcta ccatgggtac cgagtacaag cccacggtgc gcctcgccac
     3841 ccgcgacgac gtcccccggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     3901 cacgcgccac accgtcgacc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     3961 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     4021 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     4081 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     4141 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gcgtctcgcc
     4201 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     4261 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     4321 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     4381 catgacccgc aagcccggtg cctgacgccc gccccacgac ccgcagcgcc cgaccgaaag
     4441 gagcgcacga ccccatggct ccgaccgaag ccacccgggg cggccccgcc gaccccgcac
     4501 ccgcccccga ggcccaccga ctctagataa ctgatcataa tcagccatac cacatttgta
     4561 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     4621 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4681 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4741 aaactcatca atgtatctta acgctcgagt gcattctagt tgtggtttgt ccaaactcat
     4801 caatgtatct tatcatgtct gtataccgtc gacctctagc tgcaggaatt cgatatcaag
     4861 ctctagccaa caagctcgtc atcgctttgc agaagagcag agaggatatg ctcatcgtct
     4921 aaagaactac ccattttatt atatattagt cacgatatct ataacaagaa aatatatata
     4981 taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta tgagttaaat
     5041 cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc gttatagttc
     5101 aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca agcggcgact
     5161 gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga gcaatatttc
     5221 aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaaaaaa gatttgcgct
     5281 ttactcgacc taaactttaa acacgtcata gaatcttcgt ttgacaaaaa ccacattgtg
     5341 gccaagctgt gtgacgcgac gcgcgctaaa gaatggcaaa ccaagtcgcg cgaggtacca
     5401 gcttttgttc cctttagtga gggttaattt cgagcttggc gtaatcatgg tcatagctgt
     5461 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5521 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5581 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5641 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5701 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5761 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5821 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5881 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5941 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6001 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6061 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6121 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6181 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6241 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6301 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6361 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6421 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6481 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6541 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6601 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6661 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6721 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6781 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6841 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6901 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6961 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7021 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7081 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7141 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7201 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7261 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7321 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7381 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7441 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7501 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7561 aaataggggt tccgcgcaca tttccccgaa aagtgccacc t
//
