LOCUS       sequence-403732-        6500 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GST-CDK5RAP3 in E.coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6500)
  AUTHORS   Ishimura R, El-Gowily AH, Noshiro D, Komatsu-Hirota S, Ono Y, Shindo
            M, Hatta T, Abe M, Uemura T, Lee-Okada HC, Mohamed TM, Yokomizo T, 
            Ueno T, Sakimura K, Natsume T, Sorimachi H, Inada T, Waguri S, Noda 
            NN, Komatsu M
  TITLE     The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.
  JOURNAL   Nat Commun. 2022 Dec 21;13(1):7857. doi: 10.1038/s41467-022-35501-0.
  PUBMED    36543799
REFERENCE   2  (bases 1 to 6500)
  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"; journalName: "Nat 
            Commun."; date: "2022-12-21"; volume: "13(1):7857. doi"; pages: " 
            10.1038/s41467-022-35501-0"
COMMENT     Sequence Label: pGEX6p-1-CDK5RAP3
FEATURES             Location/Qualifiers
     source          1..6500
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(200..205,206..221,222..228)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    236..252
                     /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             275..928
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     886..908
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             935..958
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(2567..2589)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2734..2752)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2820..2924
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2925..2993,2994..3785)
                     /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(3143..3162)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3956..4544
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4445..4464
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4698..4715
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        4788..4865
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4866..5948
                     /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(4885..4904)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(5997..6002,6003..6020,6021..6027)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6035..6051
                     /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     6059..6075
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6059..6075
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(6071..6500,1..98)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(6091..6108)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6091..6107)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6100..6122)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgatggcgt tggaattagc ttatcgactg cacggtgcac caatgcttct ggcgtcaggc
       61 agccatcgga agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc
      121 tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca
      181 aatattctga aatgagctgt tgacaattaa tcatcggctc gtataatgtg tggaattgtg
      241 agcggataac aatttcacac aggaaacagt attcatgtcc cctatactag gttattggaa
      301 aattaagggc cttgtgcaac ccactcgact tcttttggaa tatcttgaag aaaaatatga
      361 agagcatttg tatgagcgcg atgaaggtga taaatggcga aacaaaaagt ttgaattggg
      421 tttggagttt cccaatcttc cttattatat tgatggtgat gttaaattaa cacagtctat
      481 ggccatcata cgttatatag ctgacaagca caacatgttg ggtggttgtc caaaagagcg
      541 tgcagagatt tcaatgcttg aaggagcggt tttggatatt agatacggtg tttcgagaat
      601 tgcatatagt aaagactttg aaactctcaa agttgatttt cttagcaagc tacctgaaat
      661 gctgaaaatg ttcgaagatc gtttatgtca taaaacatat ttaaatggtg atcatgtaac
      721 ccatcctgac ttcatgttgt atgacgctct tgatgttgtt ttatacatgg acccaatgtg
      781 cctggatgcg ttcccaaaat tagtttgttt taaaaaacgt attgaagcta tcccacaaat
      841 tgataagtac ttgaaatcca gcaagtatat agcatggcct ttgcagggct ggcaagccac
      901 gtttggtggt ggcgaccatc ctccaaaatc ggatctggaa gttctgttcc aggggcccct
      961 gggatccccg aattcccggg tcgacatgga ggaccatcag cacgtgccca tcgacatcca
     1021 gaccagcaag ctgctcgatt ggctggtgga cagaaggcac tgcagcctga aatggcagag
     1081 tctggtgctg acgatccgcg agaagatcaa tgctgccatc caggacatgc cagagagcga
     1141 agagatcgcc cagctgctgt ctgggtccta cattcactac tttcactgcc taagaatcct
     1201 ggaccttctc aaaggcacag aggcctccac gaagaatatt tttggccgat actcttcaca
     1261 gcggatgaag gattggcagg agattatagc tctgtatgag aaggacaaca cctacttagt
     1321 ggaactctct agcctcctgg ttcggaatgt caactatgag atcccctcac tgaagaagca
     1381 gattgccaag tgccagcagc tgcagcaaga atacagccgc aaggaggagg agtgccaggc
     1441 aggggctgcc gagatgcggg agcagttcta ccactcctgc aagcagtatg gcatcacggg
     1501 cgaaaatgtc cgaggagaac tgctggccct ggtgaaggac ctgccgagtc agctggctga
     1561 gattggggca gcggctcagc agtccctggg ggaagccatt gacgtgtacc aggcgtctgt
     1621 ggggtttgtg tgtgagagcc ccacagagca ggtgttgcca atgctgcggt tcgtgcagaa
     1681 gcggggaaac tcaacggtgt acgagtggag gacagggaca gagccctctg tggtggaacg
     1741 accccacctc gaggagcttc ctgagcaggt ggcagaagat gcgattgact ggggcgactt
     1801 tggggtagag gcagtgtctg aggggactga ctctggcatc tctgccgagg ctgctggaat
     1861 cgactggggc atcttcccgg aatcagattc aaaggatcct ggaggtgatg ggatagactg
     1921 gggagacgat gctgttgctt tgcagatcac agtgctggaa gcaggaaccc aggctccaga
     1981 aggtgttgcc aggggcccag atgccctgac actgcttgaa tacactgaga cccggaatca
     2041 gttccttgat gagctcatgg agcttgagat cttcttagcc cagagagcag tggagttgag
     2101 tgaggaggca gatgtcctgt ctgtgagcca gttccagctg gctccagcca tcctgcaggg
     2161 ccagaccaaa gagaagatgg ttaccatggt gtcagtgctg gaggatctga ttggcaagct
     2221 taccagtctt cagctgcaac acctgtttat gatcctggcc tcaccaaggt atgtggaccg
     2281 agtgactgaa ttcctccagc aaaagctgaa gcagtcccag ctgctggctt tgaagaaaga
     2341 gctgatggtg cagaagcagc aggaggcact tgaggagcag gcggctctgg agcctaagct
     2401 ggacctgcta ctggagaaga ccaaggagct gcagaagctg attgaagctg acatctccaa
     2461 gaggtacagc gggcgccctg tgaacctgat gggaacctct ctgtgagcgg ccgcatcgtg
     2521 actgactgac gatctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     2581 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     2641 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     2701 atagcggagt gtataattct tgaagacgaa agggcctcgt gatacgccta tttttatagg
     2761 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     2821 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     2881 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     2941 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     3001 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     3061 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     3121 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
     3181 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     3241 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     3301 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     3361 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     3421 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
     3481 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     3541 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     3601 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     3661 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     3721 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     3781 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     3841 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     3901 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     3961 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4021 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4081 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4141 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4201 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     4261 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     4321 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     4381 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     4441 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     4501 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     4561 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     4621 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     4681 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     4741 attttctcct tacgcatctg tgcggtattt cacaccgcat aaattccgac accatcgaat
     4801 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     4861 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     4921 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     4981 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     5041 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     5101 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     5161 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     5221 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     5281 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     5341 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     5401 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     5461 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     5521 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     5581 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     5641 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     5701 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     5761 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     5821 tgcccgtctc actggtgaaa agaaaaacca cgctagcgcc caatacgcaa accgcctctc
     5881 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     5941 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc ccaggcttta
     6001 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     6061 ggaaacagct atgaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg
     6121 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     6181 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     6241 cgaatggcgc tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg
     6301 cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga
     6361 tgcgcccatc tacaccaacg tgacctatcc cattacggtc aatccgccgt ttgttcccac
     6421 ggagaatccg acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga
     6481 aggccagacg cgaattattt
//
