LOCUS       sequence-235347-        6630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of truncated Glo3 (137-375) in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6630)
  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 6630)
  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-BoCCS
FEATURES             Location/Qualifiers
     source          1..6630
                     /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             2035..2064
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     terminator      2087..2181
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2225..2884
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFIMSKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2291..2310)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2949..3035
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3128..4210)
                     /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     4172..4191
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4211..4288)
                     /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(4372..4394)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4480..4620
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4535..4554
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4635..4652)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4806..5394)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4886..4905)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5565..6356,6357..6425))
                     /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     6188..6207
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6426..6530)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6598..6616
                     /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 gcggctcgga gttataccca
     1321 tcagaattag tcttgaacgg tcaagattct tcggactcac cgttggacac cgattcagac
     1381 gcttccaggt cgacttccaa agaaaatagt gttgacgatt ttttctctaa ttggcaaaaa
     1441 ccaagctcca attccagttc taaactcaat gtaaatacag gctctctcgc cccaaaaaac
     1501 aacaccacag gcagcacacc caagaccact gtcactaaaa cgagaagttc catcttaacg
     1561 gctagcagaa agaaaccggt attaaactcg caagataaaa agaaacattc aattttgtct
     1621 tcgtcaagaa agcccactag actaaccgca aagaaagtcg acaaatcaca ggcagaagac
     1681 ttgtttgatc aatttaaaaa agaggctcaa caagaaaagg aggacgagtt cactaattcg
     1741 tcttctagca ccaaaattag acaaaatgat tacgattccc aatttatgaa taacagtaaa
     1801 ggcaacaata ataacagtat agatgatatt aatacccagc ctgatgaatt caatgatttt
     1861 ttaaatgaca cttcaaacag cttcgacaca actagaaaag aacagcaaga tactttaaca
     1921 cccaagtttg ccaaattggg atttggtatg accatgaatg acgccaatga tttggcaaag
     1981 caacaaaaag aatctcagaa aatagctcag ggaccaagat atacgcgcgg atcccatcat
     2041 catcatcacc accaccatca ccactaagct taattagctg agcttggact cctgttgata
     2101 gatccagtaa tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg
     2161 gcgtttttta ttggtgagaa tccaagctag cttggcgaga ttttcaggag ctaaggaagc
     2221 taaaatggag aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa
     2281 agaacatttt gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct
     2341 ggatattacg gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt
     2401 tattcacatt cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg caatgaaaga
     2461 cggtgagctg gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac
     2521 tgaaacgttt tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat
     2581 atattcgcaa gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat
     2641 tgagaatatg tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa
     2701 cgtggccaat atggacaact tcttcgcccc cgttttcatc atgagcaaat attatacgca
     2761 aggcgacaag gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt
     2821 ccatgtcggc agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc
     2881 gtaatttttt taaggcagtt attggtgccc ttaaacgcct ggggtaatga ctctctagct
     2941 tgaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt
     3001 gtttgtcggt gaacgctctc ctgagtagga caaatccgcc ctctagatta cgtgcagtcg
     3061 atgataagct gtcaaacatg agaattgtgc ctaatgagtg agctaactta cattaattgc
     3121 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     3181 cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca
     3241 ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca
     3301 agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg
     3361 ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa
     3421 cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa
     3481 ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg
     3541 acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat
     3601 atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca
     3661 gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt
     3721 catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg
     3781 gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa
     3841 tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga
     3901 cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt
     3961 taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa
     4021 tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct
     4081 ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca
     4141 ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta
     4201 ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc
     4261 gaaaggtttt gcaccattcg atggtgtcgg aatttcgggc agcgttgggt cctggccacg
     4321 ggtgcgcatg atctagagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     4381 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     4441 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     4501 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     4561 gtgcaccata tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg
     4621 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     4681 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     4741 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     4801 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     4861 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     4921 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     4981 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     5041 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     5101 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     5161 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     5221 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     5281 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     5341 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     5401 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     5461 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     5521 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     5581 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     5641 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     5701 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     5761 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     5821 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     5881 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     5941 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     6001 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     6061 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     6121 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     6181 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     6241 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     6301 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     6361 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     6421 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     6481 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     6541 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     6601 aggcgtatca cgaggccctt tcgtcttcac
//
