LOCUS       sequence-235257-        7215 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 7215)
  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 7215)
  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..7215
                     /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             2620..2649
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     terminator      2672..2766
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2810..3469
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFIMSKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2876..2895)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3534..3620
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3713..4795)
                     /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     4757..4776
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4796..4873)
                     /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(4957..4979)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5065..5205
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5120..5139
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5220..5237)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5391..5979)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5471..5490)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6150..6941,6942..7010))
                     /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     6773..6792
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7011..7115)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7183..7201
                     /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 tagcagaaag
     1981 aaaccggtat taaactcgca agataaaaag aaacattcaa 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 aagataaaga agtggatgaa cgcggatccc atcatcatca tcaccaccac
     2641 catcaccact aagcttaatt agctgagctt ggactcctgt tgatagatcc agtaatgacc
     2701 tcagaactcc atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt
     2761 gagaatccaa gctagcttgg cgagattttc aggagctaag gaagctaaaa tggagaaaaa
     2821 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
     2881 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
     2941 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
     3001 ccgcctgatg aatgctcatc cggaatttcg tatggcaatg aaagacggtg agctggtgat
     3061 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
     3121 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
     3181 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     3241 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     3301 caacttcttc gcccccgttt tcatcatgag caaatattat acgcaaggcg acaaggtgct
     3361 gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat
     3421 gcttaatgaa ttacaacagt actgcgatga gtggcagggc ggggcgtaat ttttttaagg
     3481 cagttattgg tgcccttaaa cgcctggggt aatgactctc tagcttgagg catcaaataa
     3541 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     3601 ctctcctgag taggacaaat ccgccctcta gattacgtgc agtcgatgat aagctgtcaa
     3661 acatgagaat tgtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     3721 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3781 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     3841 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     3901 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     3961 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     4021 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     4081 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     4141 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     4201 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     4261 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     4321 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     4381 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     4441 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     4501 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     4561 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     4621 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     4681 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     4741 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     4801 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcacc
     4861 attcgatggt gtcggaattt cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcta
     4921 gagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     4981 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     5041 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt
     5101 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     5161 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc
     5221 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     5281 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     5341 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     5401 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     5461 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     5521 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     5581 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     5641 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     5701 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     5761 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     5821 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     5881 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     5941 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     6001 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     6061 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     6121 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     6181 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     6241 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     6301 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     6361 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     6421 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     6481 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     6541 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     6601 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     6661 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     6721 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     6781 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     6841 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     6901 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     6961 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     7021 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     7081 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     7141 gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg
     7201 ccctttcgtc ttcac
//
