LOCUS       sequence-255904-        7219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Glo3 Delta-AmpH (1-459) in E.Coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7219)
  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 7219)
  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-GAP-BoCCS-GRM
FEATURES             Location/Qualifiers
     source          1..7219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    11..27
                     /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             46..57
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             73..1170
                     /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     1144..1167
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1186..1206
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2569..2598
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     terminator      2621..2715
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2825..2844)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3487..3573
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3666..4748)
                     /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     4710..4729
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4749..4826)
                     /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(4910..4932)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5018..5158
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5073..5092
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5173..5190)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5344..5932)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5424..5443)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6103..6894,6895..6963))
                     /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     6726..6745
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6964..7068)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7136..7154
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(7178..7192,7193..7198,7199..7215,7216..7219,1..3)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    7198..7214
                     /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)."
ORIGIN
        1 atagattcaa ttgtgagcgg ataacaattt cacacagaat tcattaaaga ggagaaatta
       61 accatgaccg ggaaaactga agaaggtaaa ctggtaatct ggattaacgg cgataaaggc
      121 tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag ataccggaat taaagtcacc
      181 gttgagcatc cggataaact ggaagagaaa ttcccacagg ttgcggcaac tggcgatggc
      241 cctgacatta tcttctgggc acacgaccgc tttggtggct acgctcaatc tggcctgttg
      301 gctgaaatca ccccggacaa agcgttccag gacaagctgt atccgtttac ctgggatgcc
      361 gtacgttaca acggcaagct gattgcttac ccgatcgctg ttgaagcgtt atcgctgatt
      421 tataacaaag atctgctgcc gaacccgcca aaaacctggg aagagatccc ggcgctggat
      481 aaagaactga aagcgaaagg taagagcgcg ctgatgttca acctgcaaga accgtacttc
      541 acctggccgc tgattgctgc tgacgggggt tatgcgttca agtatgaaaa cggcaagtac
      601 gacattaaag acgtgggcgt ggataacgct ggcgcgaaag cgggtctgac cttcctggtt
      661 gacctgatta aaaacaaaca catgaatgca gacactgatt actccatcgc agaagctgcc
      721 tttaataaag gcgaaacagc gatgaccatc aacggcccgt gggcatggtc caacatcgac
      781 accagcaaag tgaattatgg tgtaacggta ctgccgacct tcaagggtca accatccaaa
      841 ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca gtccgaacaa agagctggca
      901 aaagagttcc tcgaaaacta tctgctgact gatgaaggtc tggaagcggt taataaagac
      961 aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag agttggcgaa agatccacgt
     1021 attgccgcca cgatggaaaa cgcccagaaa ggtgaaatca tgccgaacat cccgcagatg
     1081 tccgctttct ggtatgccgt gcgtactgcg gtgatcaacg ccgccagcgg tcgtcagact
     1141 gtcgatgaag ccctgaaaga cgcgcagact aattcgagct cgggtgaaaa cctctacttc
     1201 caagggtcca tgggtacccg gggggaaggc ggcggcggct cgggcggcgg cggctcgatg
     1261 agtaacgatg aaggagaaac atttgccacg gaacagacta ctcagcaagt ttttcagaag
     1321 ctaggtagca acatggaaaa tcgagtttgt tttgattgtg gtaacaagaa tcctacttgg
     1381 acttcggtac cttttggtgt gatgctgtgt attcaatgct ctgctgtgca tagaaacatg
     1441 ggtgttcata tcacctttgt aaagtcttct acattagata aatggacaat taacaattta
     1501 agaaggttta agttgggcgg gaaccataag gctcgtgatt ttttcttaaa gaacaatggg
     1561 aaacagctac taaatacagc aaatgttgac gcgaaaacaa aatatacgag ccctgttgct
     1621 aagaaatata agatacatct cgacaagaag gttcaaaagg atatggagtt atacccatca
     1681 gaattagtct tgaacggtca agattcttcg gactcaccgt tggacaccga ttcagacgct
     1741 tccaggtcga cttccaaaga aaatagtgtt gacgattttt tctctaattg gcaaaaacca
     1801 agctccaatt ccagttctaa actcaatgta aatacaggct ctctcgcccc aaaaaacaac
     1861 accacaggca gcacacccaa gaccactgtc actaaaacga gaagttccat cttaacggct
     1921 agcagaaaga aaccggtatt aaactcgcaa gataaaaaga aacattcaat tttgtcttcg
     1981 tcaagaaagc ccactagact aaccgcaaag aaagtcgaca aatcacaggc agaagacttg
     2041 tttgatcaat ttaaaaaaga ggctcaacaa gaaaaggagg acgagttcac taattcgtct
     2101 tctagcacca aaattagaca aaatgattac gattcccaat ttatgaataa cagtaaaggc
     2161 aacaataata acagtataga tgatattaat acccagcctg atgaattcaa tgatttttta
     2221 aatgacactt caaacagctt cgacacaact agaaaagaac agcaagatac tttaacaccc
     2281 aagtttgcca aattgggatt tggtatgacc atgaatgacg ccaatgattt ggcaaagcaa
     2341 caaaaagaat ctcagaaaat agctcaggga ccaagatata cgggcagaat cgcggaaagg
     2401 tacggcacac aaaaagctat atcatctgat caattatttg gtcgtggctc atttgatgaa
     2461 gcagcaaaca gggaagcgca tgacaagttg aaaacatttg acaatgctac atcaatttcc
     2521 tcttcttcat actttggaga agataaagaa gtggatgaac gcggatccca tcatcatcat
     2581 caccaccacc atcaccacta agcttaatta gctgagcttg gactcctgtt gatagatcca
     2641 gtaatgacct cagaactcca tctggatttg ttcagaacgc tcggttgccg ccgggcgttt
     2701 tttattggtg agaatccaag ctagcttggc gagattttca ggagctaagg aagctaaaat
     2761 ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc gtaaagaaca
     2821 ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc agctggatat
     2881 tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg cctttattca
     2941 cattcttgcc cgcctgatga atgctcatcc ggaatttcgt atggcaatga aagacggtga
     3001 gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc aaactgaaac
     3061 gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac acatatattc
     3121 gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt ttattgagaa
     3181 tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt taaacgtggc
     3241 caatatggac aacttcttcg cccccgtttt caccatgcat gggcaaatat tatacgcaag
     3301 gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt gatggcttcc
     3361 atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag ggcggggcgt
     3421 aattttttta aggcagttat tggtgccctt aaacgcctgg ggtaatgact ctctagcttg
     3481 aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt
     3541 ttgtcggtga acgctctcct gagtaggaca aatccgccct ctagattacg tgcagtcgat
     3601 gataagctgt caaacatgag aattgtgcct aatgagtgag ctaacttaca ttaattgcgt
     3661 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3721 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc
     3781 agtgagacgg gcaacagctg attgcccttc accgcctggc cctgagagag ttgcagcaag
     3841 cggtccacgc tggtttgccc cagcaggcga aaatcctgtt tgatggtggt taacggcggg
     3901 atataacatg agctgtcttc ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg
     3961 cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc
     4021 agcatcgcag tgggaacgat gccctcattc agcatttgca tggtttgttg aaaaccggac
     4081 atggcactcc agtcgccttc ccgttccgct atcggctgaa tttgattgcg agtgagatat
     4141 ttatgccagc cagccagacg cagacgcgcc gagacagaac ttaatgggcc cgctaacagc
     4201 gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca
     4261 tgggagaaaa taatactgtt gatgggtgtc tggtcagaga catcaagaaa taacgccgga
     4321 acattagtgc aggcagcttc cacagcaatg gcatcctggt catccagcgg atagttaatg
     4381 atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg
     4441 ccgcttcgtt ctaccatcga caccaccacg ctggcaccca gttgatcggc gcgagattta
     4501 atcgccgcga caatttgcga cggcgcgtgc agggccagac tggaggtggc aacgccaatc
     4561 agcaacgact gtttgcccgc cagttgttgt gccacgcggt tgggaatgta attcagctcc
     4621 gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc
     4681 acgcgggaaa cggtctgata agagacaccg gcatactctg cgacatcgta taacgttact
     4741 ggtttcacat tcaccaccct gaattgactc tcttccgggc gctatcatgc cataccgcga
     4801 aaggttttgc accattcgat ggtgtcggaa tttcgggcag cgttgggtcc tggccacggg
     4861 tgcgcatgat ctagagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     4921 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     4981 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     5041 gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt
     5101 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     5161 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     5221 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     5281 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     5341 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     5401 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     5461 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     5521 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     5581 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     5641 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     5701 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     5761 gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt
     5821 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     5881 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     5941 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     6001 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     6061 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     6121 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     6181 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     6241 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     6301 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     6361 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     6421 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     6481 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     6541 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     6601 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     6661 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     6721 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     6781 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     6841 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     6901 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     6961 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     7021 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     7081 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     7141 gcgtatcacg aggccctttc gtcttcacct cgagaaatca taaaaaattt atttgctttg
     7201 tgagcggata acaattata
//
