LOCUS       sequence-76838-e        5551 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses RalB G23V Delta-C in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5551)
  AUTHORS   Simicek M, Lievens S, Laga M, Guzenko D, Aushev VN, Kalev P, Baietti
            MF, Strelkov SV, Gevaert K, Tavernier J, Sablina AA
  TITLE     The deubiquitylase USP33 discriminates between RALB functions in 
            autophagy and innate immune response.
  JOURNAL   Nat Cell Biol. 2013 Oct;15(10):1220-30. doi: 10.1038/ncb2847. Epub 
            2013 Sep 22.
  PUBMED    24056301
REFERENCE   2  (bases 1 to 5551)
  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"; doi: "10.1038/ncb2847"; 
            journalName: "Nat Cell Biol"; date: "2013-10"; volume: "15"; issue: 
            "10"; pages: "1220-30"
COMMENT     Sequence Label: pGEX RalB G23V Delta-C
FEATURES             Location/Qualifiers
     source          1..5551
                     /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..935
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1601..1623)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1768..1786)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1854..1958
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1959..2027,2028..2819)
                     /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(2177..2196)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2990..3578
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3479..3498
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3732..3749
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3822..3899
                     /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             3900..4982
                     /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(3919..3938)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    4995..5016
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5031..5036,5037..5054,5055..5061)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5069..5085
                     /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     5093..5109
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5093..5109
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             join(5105..5551,1..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(5125..5142)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5125..5141)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5134..5156)
                     /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 gttccgcgtg gatccgctgc caacaagagt aagggccaga
      961 gctccttggc cctccacaag gtgatcatgg ttggcagcgt aggcgttggc aagtcagccc
     1021 tgacgcttca gttcatgtat gacgagtttg tagaagacta tgaacctacc aaagctgaca
     1081 gttatagaaa gaaagtggtt cttgatgggg aagaagttca gatagatatt ctggacaccg
     1141 ctgggcaaga ggactacgca gccattcgag ataactactt tcggagtggg gaagggtttc
     1201 ttcttgtgtt ctcaatcaca gaacatgaat cctttacagc aacagccgaa ttcagggaac
     1261 agattctccg tgtgaaggct gaagaagata aaattccact gctcgtcgtg ggaaacaagt
     1321 ctgacctaga ggagcggagg caggtgcctg tggaggaggc caggagtaaa gccgaagagt
     1381 ggggcgtgca gtacgtggag acgtcagcga agacccgggc caacgtggac aaggtgttct
     1441 ttgacctaat gagagaaatc agaacaaaga agatgtcaga aaacaaagac aagaatggca
     1501 agaaaagcag caagaacaag aaaagtttta aagaactcga gcggccgcat cgtgactgac
     1561 tgacgatctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     1621 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     1681 cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg
     1741 gagtgtataa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     1801 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     1861 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1921 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     1981 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     2041 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     2101 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     2161 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc
     2221 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     2281 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     2341 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     2401 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     2461 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt
     2521 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     2581 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     2641 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     2701 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     2761 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     2821 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     2881 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     2941 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     3001 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     3061 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     3121 agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     3181 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     3241 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     3301 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     3361 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     3421 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     3481 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     3541 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     3601 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     3661 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     3721 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc
     3781 tccttacgca tctgtgcggt atttcacacc gcataaattc cgacaccatc gaatggtgca
     3841 aaacctttcg cggtatggca tgatagcgcc cggaagagag tcaattcagg gtggtgaatg
     3901 tgaaaccagt aacgttatac gatgtcgcag agtatgccgg tgtctcttat cagaccgttt
     3961 cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg
     4021 cgatggcgga gctgaattac attcccaacc gcgtggcaca acaactggcg ggcaaacagt
     4081 cgttgctgat tggcgttgcc acctccagtc tggccctgca cgcgccgtcg caaattgtcg
     4141 cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac
     4201 gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg
     4261 ggctgatcat taactatccg ctggatgacc aggatgccat tgctgtggaa gctgcctgca
     4321 ctaatgttcc ggcgttattt cttgatgtct ctgaccagac acccatcaac agtattattt
     4381 tctcccatga agacggtacg cgactgggcg tggagcatct ggtcgcattg ggtcaccagc
     4441 aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct
     4501 ggcataaata tctcactcgc aatcaaattc agccgatagc ggaacgggaa ggcgactgga
     4561 gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa tgagggcatc gttcccactg
     4621 cgatgctggt tgccaacgat cagatggcgc tgggcgcaat gcgcgccatt accgagtccg
     4681 ggctgcgcgt tggtgcggat atctcggtag tgggatacga cgataccgaa gacagctcat
     4741 gttatatccc gccgttaacc accatcaaac aggattttcg cctgctgggg caaaccagcg
     4801 tggaccgctt gctgcaactc tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg
     4861 tctcactggt gaaaagaaaa accaccctgg cgcccaatac gcaaaccgcc tctccccgcg
     4921 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     4981 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     5041 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     5101 agctatgacc atgattacgg attcactggc cgtcgtttta caacgtcgtg actgggaaaa
     5161 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     5221 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     5281 gcgctttgcc tggtttccgg caccagaagc ggtgccggaa agctggctgg agtgcgatct
     5341 tcctgaggcc gatactgtcg tcgtcccctc aaactggcag atgcacggtt acgatgcgcc
     5401 catctacacc aacgtaacct atcccattac ggtcaatccg ccgtttgttc ccacggagaa
     5461 tccgacgggt tgttactcgc tcacatttaa tgttgatgaa agctggctac aggaaggcca
     5521 gacgcgaatt atttttgatg gcgttggaat t
//
