LOCUS       sequence-255911-        6634 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 6634)
  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 6634)
  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..6634
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..19
                     /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    35..51
                     /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             70..81
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             97..1194
                     /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     1168..1191
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1210..1230
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2008..2037
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     terminator      2060..2154
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2264..2283)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2926..3012
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3105..4187)
                     /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     4149..4168
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4188..4265)
                     /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(4349..4371)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4457..4597
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4512..4531
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4612..4629)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4783..5371)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4863..4882)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5542..6333,6334..6402))
                     /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     6165..6184
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6403..6507)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6575..6593
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(6617..6631,6632..6634,1..3,4..20,21..27)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
ORIGIN
        1 ctttgtgagc ggataacaat tataatagat tcaattgtga gcggataaca atttcacaca
       61 gaattcatta aagaggagaa attaaccatg accgggaaaa ctgaagaagg taaactggta
      121 atctggatta acggcgataa aggctataac ggtctcgctg aagtcggtaa gaaattcgag
      181 aaagataccg gaattaaagt caccgttgag catccggata aactggaaga gaaattccca
      241 caggttgcgg caactggcga tggccctgac attatcttct gggcacacga ccgctttggt
      301 ggctacgctc aatctggcct gttggctgaa atcaccccgg acaaagcgtt ccaggacaag
      361 ctgtatccgt ttacctggga tgccgtacgt tacaacggca agctgattgc ttacccgatc
      421 gctgttgaag cgttatcgct gatttataac aaagatctgc tgccgaaccc gccaaaaacc
      481 tgggaagaga tcccggcgct ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg
      541 ttcaacctgc aagaaccgta cttcacctgg ccgctgattg ctgctgacgg gggttatgcg
      601 ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg gcgtggataa cgctggcgcg
      661 aaagcgggtc tgaccttcct ggttgacctg attaaaaaca aacacatgaa tgcagacact
      721 gattactcca tcgcagaagc tgcctttaat aaaggcgaaa cagcgatgac catcaacggc
      781 ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt atggtgtaac ggtactgccg
      841 accttcaagg gtcaaccatc caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc
      901 gccagtccga acaaagagct ggcaaaagag ttcctcgaaa actatctgct gactgatgaa
      961 ggtctggaag cggttaataa agacaaaccg ctgggtgccg tagcgctgaa gtcttacgag
     1021 gaagagttgg cgaaagatcc acgtattgcc gccacgatgg aaaacgccca gaaaggtgaa
     1081 atcatgccga acatcccgca gatgtccgct ttctggtatg ccgtgcgtac tgcggtgatc
     1141 aacgccgcca gcggtcgtca gactgtcgat gaagccctga aagacgcgca gactaattcg
     1201 agctcgggtg aaaacctcta cttccaaggg tccatgggta cccgggggga aggcggcggc
     1261 ggctcgggcg gcggcggctc ggagttatac ccatcagaat tagtcttgaa cggtcaagat
     1321 tcttcggact caccgttgga caccgattca gacgcttcca ggtcgacttc caaagaaaat
     1381 agtgttgacg attttttctc taattggcaa aaaccaagct ccaattccag ttctaaactc
     1441 aatgtaaata caggctctct cgccccaaaa aacaacacca caggcagcac acccaagacc
     1501 actgtcacta aaacgagaag ttccatctta acggctagca gaaagaaacc ggtattaaac
     1561 tcgcaagata aaaagaaaca ttcaattttg tcttcgtcaa gaaagcccac tagactaacc
     1621 gcaaagaaag tcgacaaatc acaggcagaa gacttgtttg atcaatttaa aaaagaggct
     1681 caacaagaaa aggaggacga gttcactaat tcgtcttcta gcaccaaaat tagacaaaat
     1741 gattacgatt cccaatttat gaataacagt aaaggcaaca ataataacag tatagatgat
     1801 attaataccc agcctgatga attcaatgat tttttaaatg acacttcaaa cagcttcgac
     1861 acaactagaa aagaacagca agatacttta acacccaagt ttgccaaatt gggatttggt
     1921 atgaccatga atgacgccaa tgatttggca aagcaacaaa aagaatctca gaaaatagct
     1981 cagggaccaa gatatacgcg cggatcccat catcatcatc accaccacca tcaccactaa
     2041 gcttaattag ctgagcttgg actcctgttg atagatccag taatgacctc agaactccat
     2101 ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaagc
     2161 tagcttggcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
     2221 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
     2281 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
     2341 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
     2401 tgctcatccg gaatttcgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
     2461 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
     2521 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
     2581 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
     2641 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
     2701 ccccgttttc accatgcatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc
     2761 tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg
     2821 aattacaaca gtactgcgat gagtggcagg gcggggcgta atttttttaa ggcagttatt
     2881 ggtgccctta aacgcctggg gtaatgactc tctagcttga ggcatcaaat aaaacgaaag
     2941 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     3001 agtaggacaa atccgccctc tagattacgt gcagtcgatg ataagctgtc aaacatgaga
     3061 attgtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     3121 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     3181 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     3241 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     3301 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     3361 gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga ctcggtaatg
     3421 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     3481 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     3541 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     3601 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     3661 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     3721 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     3781 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     3841 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     3901 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     3961 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     4021 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     4081 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     4141 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     4201 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca ccattcgatg
     4261 gtgtcggaat ttcgggcagc gttgggtcct ggccacgggt gcgcatgatc tagagctgcc
     4321 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
     4381 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
     4441 ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga gtgtatactg
     4501 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat
     4561 accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt cctcgctcac
     4621 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4681 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4741 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4801 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4861 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4921 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4981 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5041 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5101 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5161 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5221 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5281 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5341 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5401 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5461 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5521 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5581 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5641 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5701 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5761 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5821 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5881 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5941 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6001 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6061 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6121 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6181 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6241 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6301 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6361 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6421 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6481 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     6541 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     6601 tcttcacctc gagaaatcat aaaaaattta tttg
//
