LOCUS       sequence-255882-        6334 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of truncated Glo3 (296-459) in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6334)
  AUTHORS   Arakel EC, Huranova M, Estrada AF, Rau EM, Spang A, Schwappach B
  TITLE     Dissection of GTPase-activating proteins reveals functional 
            asymmetry in the COPI coat of budding yeast.
  JOURNAL   J Cell Sci. 2019 Aug 29;132(16). pii: jcs.232124. doi: 
            10.1242/jcs.232124.
  PUBMED    31331965
REFERENCE   2  (bases 1 to 6334)
  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: "J Cell 
            Sci."; date: "2019-08-29"; volume: "132(16). pii: jcs.232124. doi"; 
            pages: " 10.1242/jcs.232124"
COMMENT     Sequence Label: A102-Par-BoCCS-GRM
FEATURES             Location/Qualifiers
     source          1..6334
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..15,16..21,22..38,39..45)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    21..37
                     /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)."
     protein_bind    53..69
                     /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)."
     RBS             88..99
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             115..1212
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     1186..1209
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1228..1248
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1726..1755
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     terminator      1778..1872
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(1982..2001)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2644..2730
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2823..3905)
                     /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     3867..3886
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3906..3983)
                     /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."
     primer_bind     complement(4067..4089)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4175..4315
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4230..4249
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4330..4347)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4501..5089)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4581..4600)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5260..6051,6052..6120))
                     /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     5883..5902
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6121..6225)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6293..6311
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcataaaaaa tttatttgct ttgtgagcgg ataacaatta taatagattc aattgtgagc
       61 ggataacaat ttcacacaga attcattaaa gaggagaaat taaccatgac cgggaaaact
      121 gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa
      181 gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa
      241 ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg
      301 gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac
      361 aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag
      421 ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg
      481 ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa
      541 ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct
      601 gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc
      661 gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa
      721 cacatgaatg cagacactga ttactccatc gcagaagctg cctttaataa aggcgaaaca
      781 gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat
      841 ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg
      901 agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac
      961 tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta
     1021 gcgctgaagt cttacgagga agagttggcg aaagatccac gtattgccgc cacgatggaa
     1081 aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc
     1141 gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa
     1201 gacgcgcaga ctaattcgag ctcgggtgaa aacctctact tccaagggtc catgggtacc
     1261 cggggggaag gcggcggcgg ctcgggcggc ggcggctcga tgaataacag taaaggcaac
     1321 aataataaca gtatagatga tattaatacc cagcctgatg aattcaatga ttttttaaat
     1381 gacacttcaa acagcttcga cacaactaga aaagaacagc aagatacttt aacacccaag
     1441 tttgccaaat tgggatttgg tatgaccatg aatgacgcca atgatttggc aaagcaacaa
     1501 aaagaatctc agaaaatagc tcagggacca agatatacgg gcagaatcgc ggaaaggtac
     1561 ggcacacaaa aagctatatc atctgatcaa ttatttggtc gtggctcatt tgatgaagca
     1621 gcaaacaggg aagcgcatga caagttgaaa acatttgaca atgctacatc aatttcctct
     1681 tcttcatact ttggagaaga taaagaagtg gatgaacgcg gatcccatca tcatcatcac
     1741 caccaccatc accactaagc ttaattagct gagcttggac tcctgttgat agatccagta
     1801 atgacctcag aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt
     1861 attggtgaga atccaagcta gcttggcgag attttcagga gctaaggaag ctaaaatgga
     1921 gaaaaaaatc actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt
     1981 tgaggcattt cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac
     2041 ggccttttta aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat
     2101 tcttgcccgc ctgatgaatg ctcatccgga atttcgtatg gcaatgaaag acggtgagct
     2161 ggtgatatgg gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt
     2221 ttcatcgctc tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca
     2281 agatgtggcg tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat
     2341 gtttttcgtc tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa
     2401 tatggacaac ttcttcgccc ccgttttcac catgcatggg caaatattat acgcaaggcg
     2461 acaaggtgct gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg
     2521 tcggcagaat gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaat
     2581 ttttttaagg cagttattgg tgcccttaaa cgcctggggt aatgactctc tagcttgagg
     2641 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
     2701 tcggtgaacg ctctcctgag taggacaaat ccgccctcta gattacgtgc agtcgatgat
     2761 aagctgtcaa acatgagaat tgtgcctaat gagtgagcta acttacatta attgcgttgc
     2821 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     2881 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     2941 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     3001 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     3061 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     3121 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     3181 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     3241 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     3301 tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg
     3361 atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg
     3421 gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca
     3481 ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc
     3541 agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg
     3601 cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc
     3661 gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc
     3721 aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc
     3781 atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg
     3841 cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt
     3901 ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag
     3961 gttttgcacc attcgatggt gtcggaattt cgggcagcgt tgggtcctgg ccacgggtgc
     4021 gcatgatcta gagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     4081 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     4141 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     4201 atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     4261 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc
     4321 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     4381 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     4441 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4501 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4561 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4621 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4681 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4741 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4801 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4861 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4921 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     4981 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     5041 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     5101 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     5161 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     5221 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     5281 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     5341 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     5401 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     5461 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     5521 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     5581 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5641 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5701 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5761 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5821 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5881 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5941 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     6001 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     6061 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     6121 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     6181 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     6241 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     6301 tatcacgagg ccctttcgtc ttcacctcga gaaa
//
