LOCUS       sequence-464208-        7526 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  High expression of the 
            Clusterin(A108E,L109S,E112K,P115Q,K304A,E307H, p.Arg214_Leu238del) 
            mutant for purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7526)
  TITLE     Hartl lab plasmids 2024
REFERENCE   2  (bases 1 to 7526)
  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 DD1 + TL1
FEATURES             Location/Qualifiers
     source          1..7526
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(1..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(2934..2951)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2940..3164
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3448..3495
                     /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      3589..3723
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3728..4330
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MGTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIE
                     RVTELQELFLTRVGLDIGKVWVADDGAAVEVWTTPESVEAGAVFAEIGPRMAELSGSRL
                     AAQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLET
                     SAPRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     4227..4247
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    4564..4685
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4601..4620)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5153..5187
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(5336..5354)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5375..5391)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5375..5391)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5388..5410)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5399..5415
                     /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(5423..5429,5430..5447,5448..5453))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5468..5489
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5606..5623)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5777..6365)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5857..5876)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6536..7327,7328..7396))
                     /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     7159..7178
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7397..7501)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aaattgtaaa cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa atcagctcac
       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 ctcggtactc
      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 tctcgagttc 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 atgatggaaa gctgggaaaa gtgtaagcaa tgcctgaaac
     1921 agacctgcat gaagttctac gcacgcgtct gcagaagtgg ctcaggcctg gttggccgcc
     1981 agcttgagga gttcctgaac cagagctcgc ccttctactt ctggatgaat ggtgaccgca
     2041 tcgactccct gctggagaac gaccggcagc agacgcacat gctggatgtc atgcaggacc
     2101 acttcagccg cgcgtccagc atcatagacg agctcttcca ggacaggttc ttcacccggg
     2161 agccccagga tacctaccac tacctgccct tcagcctgcc ccacaacttc cacgccatgt
     2221 tccagccctt ccttgagatg atacacgagg ctcagcaggc catggacatc cacttccata
     2281 gcccggcctt ccagcacccg ccaacagaat tcatacgaga aggcgacgat gaccggactg
     2341 tgtgccggga gatccgccac aactccacgg gctgcctgcg gatgaaggac cagtgtgacg
     2401 cgtgccggca catcttgtct gtggactgtt ccaccaacaa cccctcccag gctaagctgc
     2461 ggcgggagct cgacgaatcc ctccaggtcg ctgagaggtt gaccaggaaa tacaacgagc
     2521 tgctaaagtc ctaccagtgg aagatgctca acacctcctc cttgctggag cagctgaacg
     2581 agcagtttaa ctgggtgtcc cggctggcaa acctcacgca aggcgaagac cagtactatc
     2641 tgcgggtcac cacggtggct tcccacactt ctgactcgga cgttccttcc ggtgtcactg
     2701 aggtggtcgt gaagctcttt gactctgatc ccatcactgt gacggtccct gtagaagtct
     2761 ccaggaagaa ccctaaattt atggagaccg tggcggagaa agcgctgcag gaataccgca
     2821 aaaagcaccg ggaggagtga gcggccgcat agataactga tccagtgtgc tgggctacga
     2881 tgcatgggcc ccctaggtcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     2941 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     3001 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     3061 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     3121 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     3181 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     3241 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3301 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3361 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3421 tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga
     3481 aagtatagga acttccttgg ccaaaaagac cggtaccagg caggcagaag tatgcaaagc
     3541 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3601 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3661 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3721 ggctaccatg ggtaccgagt acaagcccac ggtgcgcctc gccacccgcg acgacgtccc
     3781 ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc gccacaccgt
     3841 cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt
     3901 cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg aggtctggac
     3961 cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc gcatggccga
     4021 gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg
     4081 gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc accagggcaa
     4141 gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc
     4201 cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc tcggcttcac
     4261 cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc
     4321 cggtgcctga cgcccgcccc acgacccgca gcgcccgacc gaaaggagcg cacgacccca
     4381 tggctccgac cgaagccacc cggggcggcc ccgccgaccc cgcacccgcc cccgaggccc
     4441 accgactcta gataactgat cataatcagc cataccacat ttgtagaggt tttacttgct
     4501 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     4561 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     4621 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     4681 tcttaacgct cgagtgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4741 tgtctgtata ccgtcgacct ctagctgcag gaattcgata tcaagctcta gccaacaagc
     4801 tcgtcatcgc tttgcagaag agcagagagg atatgctcat cgtctaaaga actacccatt
     4861 ttattatata ttagtcacga tatctataac aagaaaatat atatataata agttatcacg
     4921 taagtagaac atgaaataac aatataatta tcgtatgagt taaatcttaa aagtcacgta
     4981 aaagataatc atgcgtcatt ttgactcacg cggtcgttat agttcaaaat cagtgacact
     5041 taccgcattg acaagcacgc ctcacgggag ctccaagcgg cgactgagat gtcctaaatg
     5101 cacagcgacg gattcgcgct atttagaaag agagagcaat atttcaagaa tgcatgcgtc
     5161 aattttacgc agactatctt tctagggtta aaaaagattt gcgctttact cgacctaaac
     5221 tttaaacacg tcatagaatc ttcgtttgac aaaaaccaca ttgtggccaa gctgtgtgac
     5281 gcgacgcgcg ctaaagaatg gcaaaccaag tcgcgcgagg taccagcttt tgttcccttt
     5341 agtgagggtt aatttcgagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5401 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5461 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5521 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5581 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5641 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5701 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5761 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5821 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5881 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5941 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6001 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6061 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6121 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6181 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6241 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6301 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6361 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6421 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6481 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6541 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6601 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6661 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6721 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6781 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6841 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6901 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6961 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7021 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7081 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7141 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7201 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7261 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7321 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7381 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7441 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7501 gcacatttcc ccgaaaagtg ccacct
//
