LOCUS       sequence-256280-        7219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Glo3 Delta-AmpH lacking the COPI-binding motif 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 Delta-AmpH Delta-2x+ve
FEATURES             Location/Qualifiers
     source          1..7219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     21..39
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(63..77,78..83,84..100,101..107)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    83..99
                     /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    115..131
                     /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             150..161
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             177..1274
                     /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     1248..1271
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             1290..1310
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2673..2702
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     terminator      2725..2819
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     primer_bind     complement(2929..2948)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3591..3677
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3770..4852)
                     /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     4814..4833
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4853..4930)
                     /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(5014..5036)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5122..5262
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5177..5196
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5277..5294)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5448..6036)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5528..5547)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6207..6998,6999..7067))
                     /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     6830..6849
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7068..7172)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt cacctcgaga
       61 aatcataaaa aatttatttg ctttgtgagc ggataacaat tataatagat tcaattgtga
      121 gcggataaca atttcacaca gaattcatta aagaggagaa attaaccatg accgggaaaa
      181 ctgaagaagg taaactggta atctggatta acggcgataa aggctataac ggtctcgctg
      241 aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata
      301 aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac attatcttct
      361 gggcacacga ccgctttggt ggctacgctc aatctggcct gttggctgaa atcaccccgg
      421 acaaagcgtt ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca
      481 agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc
      541 tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga
      601 aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg
      661 ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt aaagacgtgg
      721 gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct ggttgacctg attaaaaaca
      781 aacacatgaa tgcagacact gattactcca tcgcagaagc tgcctttaat aaaggcgaaa
      841 cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt
      901 atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc
      961 tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcaaaagag ttcctcgaaa
     1021 actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg ctgggtgccg
     1081 tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc acgtattgcc gccacgatgg
     1141 aaaacgccca gaaaggtgaa atcatgccga acatcccgca gatgtccgct ttctggtatg
     1201 ccgtgcgtac tgcggtgatc aacgccgcca gcggtcgtca gactgtcgat gaagccctga
     1261 aagacgcgca gactaattcg agctcgggtg aaaacctcta cttccaaggg tccatgggta
     1321 cccgggggga aggcggcggc ggctcgggcg gcggcggctc gatgagtaac gatgaaggag
     1381 aaacatttgc cacggaacag actactcaac aagtctttca gaagctaggt agcaacatgg
     1441 aaaatcgagt ttgttttgat tgtggtaaca agaatcctac ttggacttcg gtaccttttg
     1501 gtgtgatgct gtgtattcaa tgctctgctg tgcatagaaa catgggtgtt catatcacct
     1561 ttgtaaagtc ttctacatta gataaatgga caattaacaa tttaagaagg tttaagttgg
     1621 gcgggaacca taaggctcgt gattttttct taaagaacaa tgggaaacag ctactaaata
     1681 cagcaaatgt tgacgcgaaa acaaaatata cgagccctgt tgctaagaaa tataagatac
     1741 atctcgacaa gaaggttcaa aaggatatgg agttataccc atcagaatta gtcttgaacg
     1801 gtcaagattc ttcggactca ccgttggaca ccgattcaga cgcttccagg tcgacttcca
     1861 aagaaaatag tgttgacgat tttttctcta attggcaaaa accaagctcc aattccagtt
     1921 ctaaactcaa tgtaaataca ggctctctcg ccccaaaaaa caacaccaca ggcagcacac
     1981 ccaagaccac tgtcactaaa acgagaagtt ctatcttaac ggctagcgaa gaggaaccgg
     2041 tattaaactc gcaagatgaa gaggaacatt caattttgtc ttcgtcaaga aagcccacta
     2101 gactaaccgc aaagaaagtc gacaaatcac aggcagaaga cttgtttgat caatttaaaa
     2161 aagaggctca acaagaaaag gaggacgagt tcactaattc gtcttctagc accaaaatta
     2221 gacaaaatga ttacgattcc caatttatga ataacagtaa aggcaacaat aataacagta
     2281 tagatgatat taatacccag cctgatgaat tcaatgattt tttaaatgac acttcaaaca
     2341 gcttcgacac aactagaaaa gaacagcaag atactttaac acccaagttt gccaaattgg
     2401 gatttggtat gaccatgaat gacgccaatg atttggcaaa gcaacaaaaa gaatctcaga
     2461 aaatagctca gggaccaaga tatacgggca gaatcgcgga aaggtacggc acacaaaaag
     2521 ctatatcatc tgatcaatta tttggtcgtg gctcatttga tgaagcagca aacagggaag
     2581 cgcatgacaa gttgaaaaca tttgacaatg ctacatcaat ttcctcttct tcatactttg
     2641 gagaagataa agaagtggat gaacgcggat cccatcatca tcatcaccac caccatcacc
     2701 actaagctta attagctgag cttggactcc tgttgataga tccagtaatg acctcagaac
     2761 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc
     2821 caagctagct tggcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact
     2881 ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag
     2941 tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag
     3001 accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg
     3061 atgaatgctc atccggaatt tcgtatggca atgaaagacg gtgagctggt gatatgggat
     3121 agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg
     3181 agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt
     3241 tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca
     3301 gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc
     3361 ttcgcccccg ttttcaccat gcatgggcaa atattatacg caaggcgaca aggtgctgat
     3421 gccgctggcg attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct
     3481 taatgaatta caacagtact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag
     3541 ttattggtgc ccttaaacgc ctggggtaat gactctctag cttgaggcat caaataaaac
     3601 gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc
     3661 tcctgagtag gacaaatccg ccctctagat tacgtgcagt cgatgataag ctgtcaaaca
     3721 tgagaattgt gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc
     3781 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3841 aggcggtttg cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca
     3901 gctgattgcc cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt
     3961 gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt
     4021 cttcggtatc gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg
     4081 taatggcgcg cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa
     4141 cgatgccctc attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc
     4201 cttcccgttc cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca
     4261 gacgcagacg cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac
     4321 ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac
     4381 tgttgatggg tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag
     4441 cttccacagc aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc
     4501 gttgcgcgag aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca
     4561 tcgacaccac cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt
     4621 gcgacggcgc gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc
     4681 ccgccagttg ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca
     4741 ctttttcccg cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct
     4801 gataagagac accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca
     4861 ccctgaattg actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcaccatt
     4921 cgatggtgtc ggaatttcgg gcagcgttgg gtcctggcca cgggtgcgca tgatctagag
     4981 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     5041 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     5101 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     5161 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt
     5221 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg
     5281 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     5341 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     5401 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     5461 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     5521 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     5581 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     5641 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     5701 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     5761 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     5821 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     5881 actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     5941 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     6001 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     6061 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     6121 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     6181 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     6241 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     6301 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     6361 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     6421 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     6481 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     6541 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     6601 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     6661 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     6721 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     6781 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     6841 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     6901 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     6961 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     7021 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     7081 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     7141 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     7201 gtctaagaaa ccattatta
//
