LOCUS       sequence-235348-        6099 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of truncated Glo3 (373-459) in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6099)
  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 6099)
  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-GRM
FEATURES             Location/Qualifiers
     source          1..6099
                     /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             1504..1533
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     terminator      1556..1650
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1694..2353
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFIMSKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1760..1779)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2418..2504
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2597..3679)
                     /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     3641..3660
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3680..3757)
                     /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(3841..3863)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3949..4089
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4004..4023
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4104..4121)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4275..4863)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4355..4374)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5034..5825,5826..5894))
                     /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     5657..5676
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5895..5999)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6067..6085
                     /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 gcggctcgag atatacgggc
     1321 agaatcgcgg aaaggtacgg cacacaaaaa gctatatcat ctgatcaatt atttggtcgt
     1381 ggctcatttg atgaagcagc aaacagggaa gcgcatgaca agttgaaaac atttgacaat
     1441 gctacatcaa tttcctcttc ttcatacttt ggagaagata aagaagtgga tgaacgcgga
     1501 tcccatcatc atcatcacca ccaccatcac cactaagctt aattagctga gcttggactc
     1561 ctgttgatag atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt
     1621 tgccgccggg cgttttttat tggtgagaat ccaagctagc ttggcgagat tttcaggagc
     1681 taaggaagct aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg
     1741 gcatcgtaaa gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac
     1801 cgttcagctg gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta
     1861 tccggccttt attcacattc ttgcccgcct gatgaatgct catccggaat ttcgtatggc
     1921 aatgaaagac ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca
     1981 tgagcaaact gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt
     2041 tctacacata tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa
     2101 agggtttatt gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt
     2161 tgatttaaac gtggccaata tggacaactt cttcgccccc gttttcatca tgagcaaata
     2221 ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg
     2281 tgatggcttc catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca
     2341 gggcggggcg taattttttt aaggcagtta ttggtgccct taaacgcctg gggtaatgac
     2401 tctctagctt gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt
     2461 ttatctgttg tttgtcggtg aacgctctcc tgagtaggac aaatccgccc tctagattac
     2521 gtgcagtcga tgataagctg tcaaacatga gaattgtgcc taatgagtga gctaacttac
     2581 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2641 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     2701 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     2761 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     2821 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     2881 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     2941 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     3001 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     3061 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     3121 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     3181 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     3241 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     3301 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     3361 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     3421 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     3481 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     3541 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     3601 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     3661 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     3721 ccataccgcg aaaggttttg caccattcga tggtgtcgga atttcgggca gcgttgggtc
     3781 ctggccacgg gtgcgcatga tctagagctg cctcgcgcgt ttcggtgatg acggtgaaaa
     3841 cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag
     3901 cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac
     3961 ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt
     4021 gtactgagag tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     4081 cgcatcaggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4141 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4201 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4261 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4321 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4381 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4441 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4501 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4561 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4621 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4681 ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
     4741 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4801 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4861 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4921 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4981 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5041 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5101 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5161 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5221 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5281 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5341 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5401 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5461 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5521 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5581 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5641 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5701 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5761 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5821 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5881 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     5941 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6001 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     6061 tataaaaata ggcgtatcac gaggcccttt cgtcttcac
//
