LOCUS       sequence-145746-        5737 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses Sacsin sr1 (SIRPT1 repeat) domain (84-339) in fusion with 
            GST (bacterial).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5737)
  AUTHORS   Menade M, Kozlov G, Trempe JF, Pande H, Shenker S, Wickremasinghe S,
            Li X, Hojjat H, Dicaire MJ, Brais B, McPherson PS, Wong MJH, Young 
            JC, Gehring K
  TITLE     Structures of ubiquitin-like (Ubl) and Hsp90-like domains of sacsin 
            provide insight into pathological mutations.
  JOURNAL   J Biol Chem. 2018 Aug 17;293(33):12832-12842. doi: 
            10.1074/jbc.RA118.003939. Epub 2018 Jun 26.
  PUBMED    29945973
REFERENCE   2  (bases 1 to 5737)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1074/jbc.RA118.003939"; journalName: "J Biol Chem"; date: 
            "2018-08-17- 17"; volume: "293"; issue: "33"; pages: "12832-12842"
COMMENT     Sequence Label: pGEX6P1-sr1(SIRPT1)
FEATURES             Location/Qualifiers
     source          1..5737
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(183..188,189..204,205..211)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    219..235
                     /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)."
     CDS             258..911
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     869..891
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             918..941
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1787..1809)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1954..1972)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2040..2144
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2145..2213,2214..3005)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2363..2382)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3176..3764
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3665..3684
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3918..3935
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4008..4085
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             4086..5168
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPSTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(4105..4124)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    5181..5202
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5217..5222,5223..5240,5241..5247)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5255..5271
                     /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)."
     primer_bind     5279..5295
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5279..5295
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5291..5737,1..81)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(5311..5328)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5311..5327)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5320..5342)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 acgttatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc ggaagctgtg
       61 gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc gcactcccgt
      121 tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc tgaaatgagc
      181 tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat aacaatttca
      241 cacaggaaac agtattcatg tcccctatac taggttattg gaaaattaag ggccttgtgc
      301 aacccactcg acttcttttg gaatatcttg aagaaaaata tgaagagcat ttgtatgagc
      361 gcgatgaagg tgataaatgg cgaaacaaaa agtttgaatt gggtttggag tttcccaatc
      421 ttccttatta tattgatggt gatgttaaat taacacagtc tatggccatc atacgttata
      481 tagctgacaa gcacaacatg ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc
      541 ttgaaggagc ggttttggat attagatacg gtgtttcgag aattgcatat agtaaagact
      601 ttgaaactct caaagttgat tttcttagca agctacctga aatgctgaaa atgttcgaag
      661 atcgtttatg tcataaaaca tatttaaatg gtgatcatgt aacccatcct gacttcatgt
      721 tgtatgacgc tcttgatgtt gttttataca tggacccaat gtgcctggat gcgttcccaa
      781 aattagtttg ttttaaaaaa cgtattgaag ctatcccaca aattgataag tacttgaaat
      841 ccagcaagta tatagcatgg cctttgcagg gctggcaagc cacgtttggt ggtggcgacc
      901 atcctccaaa atcggatctg gaagttctgt tccaggggcc cctgggatcc aaagggggag
      961 gtcgatttgg tcagacaacg ccaccacttg ttgattttct caaggacatt ttgagaagat
     1021 atccagaagg aggacagatt cttaaggaat taattcagaa tgcagaagat gctggggcga
     1081 cagaagttaa atttttatat gatgaaactc aatacggaac agagactctt tggtcaaaag
     1141 atatggcgcc atatcagggg ccagctctct atgtgtacaa caacgcggtt ttcaccccag
     1201 aggactggca cggcattcaa gaaatagcaa gaagcaggaa aaaggatgat cctctgaagg
     1261 tcggaagatt tggaattggg tttaattctg tctatcatat aacagatgtt ccttgtatct
     1321 ttagtggtga ccaaatcggg atgctagatc ctcatcaaac actttttggc ccacatgaat
     1381 caggccaatg ttggaatctc aaagatgaca gcaaagaaat tagtgaactt tcagaccagt
     1441 ttgcaccatt tgttggcatt tttggaagca ccaaggaaac atttataaac ggcaattttc
     1501 caggaacatt tttccgtttc cctcttcgcc tacaaccttc acaacttagt agtaacctct
     1561 acaataagca gaaggttctt gagttgtttg agtcttttag ggcagatgcg gacacagtgc
     1621 tgctctttct gaaaagtgtg caggatgttt ccttatatgt ccgagaggct gacggaacag
     1681 agaaactggt gtttagagtg acttcgagtg agagtaagta gctcgagcgg ccgcatcgtg
     1741 actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     1801 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     1861 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     1921 atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     1981 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2041 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2101 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2161 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     2221 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     2281 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     2341 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     2401 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     2461 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     2521 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     2581 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     2641 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     2701 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     2761 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     2821 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     2881 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     2941 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3001 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3061 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3121 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3181 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     3241 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3301 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3361 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3421 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3481 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3541 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3601 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3661 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3721 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3781 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3841 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3901 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     3961 attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac accatcgaat
     4021 ggtgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     4081 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     4141 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     4201 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     4261 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     4321 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     4381 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     4441 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     4501 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     4561 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     4621 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     4681 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     4741 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     4801 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     4861 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     4921 gctcatgtta tatcccgccg tcaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     4981 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     5041 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     5101 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     5161 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     5221 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     5281 ggaaacagct atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg
     5341 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     5401 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     5461 cgaatggcgc tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg
     5521 cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga
     5581 tgcgcccatc tacaccaacg taacctatcc cattacggtc aatccgccgt ttgttcccac
     5641 ggagaatccg acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga
     5701 aggccagacg cgaattattt ttgatggcgt tggaatt
//
