LOCUS       sequence-248551-        7203 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 7203)
  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 7203)
  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..7203
                     /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             2608..2637
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     terminator      2660..2754
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2798..3457
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFIMSKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2864..2883)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3522..3608
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3701..4783)
                     /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     4745..4764
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4784..4861)
                     /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(4945..4967)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5053..5193
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5108..5127
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5208..5225)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5379..5967)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5459..5478)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6138..6929,6930..6998))
                     /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     6761..6780
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6999..7103)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7171..7189
                     /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 gcggctcgat gagtaacgat
     1321 gaaggagaaa catttgccac ggaacagact actcaacaag tttttcagaa gctaggtagc
     1381 aacatggaaa atcgagtttg ttttgattgt ggtaacaaga atcctacttg gacttcggta
     1441 ccttttggtg tgatgctgtg tattcaatgc tctgctgtgc atagaaacat gggtgttcat
     1501 atcacctttg taaagtcttc tacattagat aaatggacaa ttaacaattt aagaaggttt
     1561 aagttgggtg ggaaccataa ggctcgtgat tttttcttaa agaacaatgg gaaacagcta
     1621 ctaaatacag caaatgttga cgcgaaaaca aaatatacga gccctgttgc taagaaatat
     1681 aagatacatc tcgacaagaa ggttcaaaag gatatggagt tatacccatc agaattagtc
     1741 ttgaacggtc aagattcttc ggactcaccg ttggacaccg attcagacgc ttccaggtcg
     1801 acttccaaag aaaatagtgt tgacgatttt ttctctaatt ggcaaaaacc aagctccaat
     1861 tccagttcta aactcaatgt aaatacaggc tctctcgccc caaaaaacaa caccacaggc
     1921 agcacaccca agaccactgt cactaaaacg agaagttcca tcttaacggc tagcgaagag
     1981 gaaccggtat taaactcgca agatgaagag gaacattcaa ttttgtcttc gtcaagaaag
     2041 cccactagac taaccgcaaa gaaagtcgac aaatcacagg cagaagactt gtttgatcaa
     2101 tttaaaaaag aggctcaaca agaaaaggag gacgagttca ctaattcgtc ttctagcacc
     2161 aaaattagac aaaatgatta cgattcccaa tttatgaata acagtaaagg caacaataat
     2221 aacagtatag atgatattaa tacccagcct gatgaattca atgatttttt aaatgacact
     2281 tcaaacagct tcgacacaac tagaaaagaa cagcaagata ctttaacacc caagtttgcc
     2341 aaattgggat ttggtatgac catgaatgac gccaatgatt tggcaaagca acaaaaagaa
     2401 tctcagaaaa tagctcaggg accaagatat acgggcagaa tcgcggaaag gtacggcaca
     2461 caaaaagcta tatcatctga tcaattattt ggtcgtggct catttgatga agcagcaaac
     2521 agggaagcgc atgacaagtt gaaaacattt gacaatgcta catcaatttc ctcttcttca
     2581 tactttggag aagataaacg cggatcccat catcatcatc accaccacca tcaccactaa
     2641 gcttaattag ctgagcttgg actcctgttg atagatccag taatgacctc agaactccat
     2701 ctggatttgt tcagaacgct cggttgccgc cgggcgtttt ttattggtga gaatccaagc
     2761 tagcttggcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
     2821 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
     2881 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
     2941 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
     3001 tgctcatccg gaatttcgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
     3061 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
     3121 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
     3181 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
     3241 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
     3301 ccccgttttc atcatgagca aatattatac gcaaggcgac aaggtgctga tgccgctggc
     3361 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
     3421 acaacagtac tgcgatgagt ggcagggcgg ggcgtaattt ttttaaggca gttattggtg
     3481 cccttaaacg cctggggtaa tgactctcta gcttgaggca tcaaataaaa cgaaaggctc
     3541 agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta
     3601 ggacaaatcc gccctctaga ttacgtgcag tcgatgataa gctgtcaaac atgagaattg
     3661 tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc
     3721 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     3781 gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc
     3841 ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca
     3901 ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat
     3961 cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc
     4021 gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct
     4081 cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt
     4141 ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac
     4201 gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga
     4261 ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg
     4321 gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag
     4381 caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga
     4441 gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca
     4501 ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg
     4561 cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt
     4621 gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc
     4681 gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga
     4741 caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt
     4801 gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcaccat tcgatggtgt
     4861 cggaatttcg ggcagcgttg ggtcctggcc acgggtgcgc atgatctaga gctgcctcgc
     4921 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     4981 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     5041 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     5101 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg
     5161 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac
     5221 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     5281 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     5341 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     5401 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     5461 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5521 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5581 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5641 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5701 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5761 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg
     5821 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5881 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5941 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     6001 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     6061 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     6121 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     6181 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     6241 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     6301 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     6361 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     6421 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6481 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6541 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6601 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6661 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6721 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6781 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6841 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6901 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6961 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     7021 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     7081 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     7141 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt
     7201 cac
//
