LOCUS       sequence-326710-        6380 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of a GST fusion of e.coli optimized Parkin with
            6 alanine linker between the GST and the inserted Parkin gene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6380)
  TITLE     Haas Lab plasmids
REFERENCE   2  (bases 1 to 6380)
  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: pGEX4T-3-6AOptParkin
FEATURES             Location/Qualifiers
     source          1..6380
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      107..695
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     596..615
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     849..866
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        939..1016
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1017..2099
                     /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
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1036..1055)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(2148..2153,2154..2171,2172..2178)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2186..2202
                     /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     2210..2226
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2210..2226
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2222..2749
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(2242..2259)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2242..2258)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2251..2273)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(2851..2856,2857..2872,2873..2879)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2887..2903
                     /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             2926..3579
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     3537..3559
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             3586..3603
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(5098..5120)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5265..5283)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5351..5455
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5456..5524,5525..6316)
                     /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(5674..5693)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
       61 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
      121 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
      181 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
      241 taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
      301 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
      361 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
      421 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
      481 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
      541 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
      601 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
      661 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
      721 ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt
      781 ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga
      841 ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc
      901 ttacgcatct gtgcggtatt tcacaccgca taaattccga caccatcgaa tggcgcaaaa
      961 cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga
     1021 aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc
     1081 gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga
     1141 tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt
     1201 tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg
     1261 cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa
     1321 gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc
     1381 tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta
     1441 atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct
     1501 cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa
     1561 tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc
     1621 ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg
     1681 ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga
     1741 tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc
     1801 tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt
     1861 atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg
     1921 accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct
     1981 cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt
     2041 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     2101 cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cccaggcttt acactttatg
     2161 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     2221 tatgaccatg attacggatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     2281 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     2341 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     2401 ctttgcctgg tttccggcac cagaagcggt gccggaaagc tggctggagt gcgatcttcc
     2461 tgaggccgat actgtcgtcg tcccctcaaa ctggcagatg cacggttacg atgcgcccat
     2521 ctacaccaac gtgacctatc ccattacggt caatccgccg tttgttccca cggagaatcc
     2581 gacgggttgt tactcgctca catttaatgt tgatgaaagc tggctacagg aaggccagac
     2641 gcgaattatt tttgatggcg ttggaattag cttatcgact gcacggtgca ccaatgcttc
     2701 tggcgtcagg cagccatcgg aagctgtggt atggctgtgc aggtcgtaaa tcactgcata
     2761 attcgtgtcg ctcaaggcgc actcccgttc tggataatgt tttttgcgcc gacatcataa
     2821 cggttctggc aaatattctg aaatgagctg ttgacaatta atcatcggct cgtataatgt
     2881 gtggaattgt gagcggataa caatttcaca caggaaacag tattcatgtc ccctatacta
     2941 ggttattgga aaattaaggg ccttgtgcaa cccactcgac ttcttttgga atatcttgaa
     3001 gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg ataaatggcg aaacaaaaag
     3061 tttgaattgg gtttggagtt tcccaatctt ccttattata ttgatggtga tgttaaatta
     3121 acacagtcta tggccatcat acgttatata gctgacaagc acaacatgtt gggtggttgt
     3181 ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg ttttggatat tagatacggt
     3241 gtttcgagaa ttgcatatag taaagacttt gaaactctca aagttgattt tcttagcaag
     3301 ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc ataaaacata tttaaatggt
     3361 gatcatgtaa cccatcctga cttcatgttg tatgacgctc ttgatgttgt tttatacatg
     3421 gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt ttaaaaaacg tattgaagct
     3481 atcccacaaa ttgataagta cttgaaatcc agcaagtata tagcatggcc tttgcagggc
     3541 tggcaagcca cgtttggtgg tggcgaccat cctccaaaat cggatctggt tccgcgtgga
     3601 tccccgaatt cccgggtcga cgctgcagct gcagcagcta tgatcgtgtt tgttcgtttc
     3661 aacagcagcc acggttttcc ggttgaggtg gacagcgata ccagcatttt ccagctgaag
     3721 gaagtggttg cgaaacgtca gggtgtgccg gcggaccaac tgcgtgttat cttcgcgggc
     3781 aaggagctgc gtaacgattg gaccgttcaa aactgcgacc tggatcagca aagcatcgtg
     3841 cacattgttc agcgtccgtg gcgtaaaggt caagaaatga acgcgaccgg tggcgacgat
     3901 ccgcgtaacg cggcgggtgg ctgcgagcgt gaaccgcaga gcctgacccg tgtggacctg
     3961 agcagcagcg ttctgccggg tgatagcgtg ggcctggcgg ttatcctgca caccgacagc
     4021 cgtaaggata gcccgccggc gggtagcccg gcgggccgta gcatttataa cagcttttac
     4081 gtgtattgca agggtccgtg ccagcgtgtg caaccgggca aactgcgtgt tcagtgcagc
     4141 acctgccgtc aagcgaccct gaccctgacc cagggtccga gctgctggga cgatgttctg
     4201 attccgaacc gtatgagcgg tgaatgccaa agcccgcact gcccgggtac cagcgcggag
     4261 ttcttcttca agtgcggcgc gcacccgacc agcgacaaag agaccagcgt ggcgctgcac
     4321 ctgattgcga ccaacagccg taacatcacc tgcattacct gcaccgatgt gcgtagcccg
     4381 gtgctggttt ttcagtgcaa cagccgtcac gttatctgcc tggattgctt ccacctgtat
     4441 tgcgtgaccc gtctgaacga ccgtcagttt gttcacgatc cgcaactggg ttatagcctg
     4501 ccgtgcgttg cgggttgccc gaacagcctg atcaaagaac tgcaccactt ccgtattctg
     4561 ggcgaggaac agtacaaccg ttatcagcaa tacggcgcgg aggaatgcgt gctgcaaatg
     4621 ggtggcgttc tgtgcccgcg tccgggttgc ggtgcgggtc tgctgccgga gccggaccag
     4681 cgtaaggtga cctgcgaagg tggcaacggc ctgggttgcg gctttgcgtt ctgccgtgag
     4741 tgcaaagaag cgtatcacga gggtgaatgc agcgcggtgt ttgaagcgag cggtaccacc
     4801 acccaggcgt accgtgttga tgagcgtgcg gcggaacaag cgcgttggga ggcggcgagc
     4861 aaggaaacca tcaagaaaac caccaagccg tgcccgcgtt gccatgtgcc ggttgaaaag
     4921 aacggtggct gcatgcacat gaaatgcccg cagccgcaat gccgtctgga gtggtgctgg
     4981 aactgcggtt gcgaatggaa ccgtgtgtgc atgggcgacc actggttcga tgtttaggcg
     5041 gccgcatcgt gactgactga cgatctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     5101 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     5161 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     5221 gtcacgtagc gatagcggag tgtataattc ttgaagacga aagggcctcg tgatacgcct
     5281 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     5341 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5401 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5461 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5521 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5581 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5641 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt
     5701 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     5761 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     5821 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     5881 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     5941 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc
     6001 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6061 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6121 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6181 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6241 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6301 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6361 acttcatttt taatttaaaa
//
