LOCUS       sequence-235346-        6330 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 6330)
  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 6330)
  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..6330
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             124..1221
                     /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     1195..1218
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1237..1257
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1735..1764
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     terminator      1787..1881
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1925..2584
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFIMSKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1991..2010)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2649..2735
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2828..3910)
                     /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     3872..3891
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3911..3988)
                     /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(4072..4094)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4180..4320
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4235..4254
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4335..4352)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4506..5094)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4586..4605)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5265..6056,6057..6125))
                     /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     5888..5907
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6126..6230)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6298..6316
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aaccatgacc
      121 gggaaaactg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt
      181 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat
      241 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt
      301 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc
      361 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac
      421 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa
      481 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg
      541 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg
      601 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa
      661 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt
      721 aaaaacaaac acatgaatgc agacactgat tactccatcg cagaagctgc ctttaataaa
      781 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa
      841 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt
      901 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc
      961 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg
     1021 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc
     1081 acgatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc
     1141 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa
     1201 gccctgaaag acgcgcagac taattcgagc tcgggtgaaa acctctactt ccaagggtcc
     1261 atgggtaccc ggggggaagg cggcggcggc tcgggcggcg gcggctcgat gaataacagt
     1321 aaaggcaaca ataataacag tatagatgat attaataccc agcctgatga attcaatgat
     1381 tttttaaatg acacttcaaa cagcttcgac acaactagaa aagaacagca agatacttta
     1441 acacccaagt ttgccaaatt gggatttggt atgaccatga atgacgccaa tgatttggca
     1501 aagcaacaaa aagaatctca gaaaatagct cagggaccaa gatatacggg cagaatcgcg
     1561 gaaaggtacg gcacacaaaa agctatatca tctgatcaat tatttggtcg tggctcattt
     1621 gatgaagcag caaacaggga agcgcatgac aagttgaaaa catttgacaa tgctacatca
     1681 atttcctctt cttcatactt tggagaagat aaagaagtgg atgaacgcgg atcccatcat
     1741 catcatcacc accaccatca ccactaagct taattagctg agcttggact cctgttgata
     1801 gatccagtaa tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg
     1861 gcgtttttta ttggtgagaa tccaagctag cttggcgaga ttttcaggag ctaaggaagc
     1921 taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa
     1981 agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct
     2041 ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt
     2101 tattcacatt cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg caatgaaaga
     2161 cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac
     2221 tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat
     2281 atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat
     2341 tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa
     2401 cgtggccaat atggacaact tcttcgcccc cgttttcatc atgagcaaat attatacgca
     2461 aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt
     2521 ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc
     2581 gtaatttttt taaggcagtt attggtgccc ttaaacgcct ggggtaatga ctctctagct
     2641 tgaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     2701 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc ctctagatta cgtgcagtcg
     2761 atgataagct gtcaaacatg agaattgtgc ctaatgagtg agctaactta cattaattgc
     2821 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     2881 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     2941 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     3001 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     3061 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     3121 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     3181 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     3241 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     3301 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     3361 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     3421 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     3481 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     3541 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     3601 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     3661 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     3721 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     3781 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     3841 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     3901 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     3961 gaaaggtttt gcaccattcg atggtgtcgg aatttcgggc agcgttgggt cctggccacg
     4021 ggtgcgcatg atctagagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     4081 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     4141 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     4201 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     4261 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     4321 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4381 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4441 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4501 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4561 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4621 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4681 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     4741 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     4801 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     4861 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     4921 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     4981 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5041 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5101 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5161 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5221 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5281 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5341 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5401 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5461 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5521 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5581 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5641 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     5701 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     5761 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     5821 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     5881 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     5941 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6001 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6061 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6121 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6181 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6241 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     6301 aggcgtatca cgaggccctt tcgtcttcac
//
