LOCUS       sequence-455976-        6249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of 10xHis-SUMO (Smt3) fusion to Sec7 domain 
            (687-885) of Big1 (ArfGEF1) for rapid nucleotide exchange and 
            activation of Arf GTPases.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6249)
  AUTHORS   Begley M, Aragon M, Baker RW
  TITLE     A structure-based mechanism for initiation of AP-3 coated vesicle 
            formation.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Dec 24;121(52):e2411974121. doi: 
            10.1073/pnas.2411974121. Epub 2024 Dec 20.
  PUBMED    39705307
REFERENCE   2  (bases 1 to 6249)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2024-12-24"; volume: "121(52):e2411974121. 
            doi"; pages: " 10.1073/pnas.2411974121. Epub 2024 Dec 20"
COMMENT     Sequence Label: MZB222
FEATURES             Location/Qualifiers
     source          1..6249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             303..332
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             1322..1339
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1392..1410)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1406..1453
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1490..1945
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1577..1596)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1787..1808
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2037..2852)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2037..2852)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2760..2779
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2844..2863
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2974..3562
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3463..3482
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3716..3733
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3748..3887
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3813..3832)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3973..3995
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3989..4180)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4989..6071)
                     /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     6033..6052
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6072..6149)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgg
      301 gccatcacca tcaccatcac catcaccatc acggcagcat gagcgatagc gaagtgaacc
      361 aggaagcgaa accggaagtg aaaccggaag tgaaaccgga aacccatatt aacctgaaag
      421 tgagcgatgg cagcagcgaa atttttttta aaattaaaaa aaccaccccg ctgcgtcgtc
      481 tgatggaagc gtttgcgaaa cgtcagggca aagaaatgga tagcctgcgt tttctgtatg
      541 atggcattcg tattcaggcg gatcagaccc cggaagatct ggatatggaa gataacgata
      601 ttattgaagc gcatcgtgaa cagattggcg gcgataaccc ggaacagttt gaagtgctga
      661 aacagcagaa agaaattatt gaacagggca ttgatctgtt taacaaaaaa ccgaaacgcg
      721 gcattcagta tctgcaggaa cagggcatgc tgggcaccac cccggaagat attgcgcagt
      781 ttctgcatca ggaagaacgc ctggatagca cccaggtggg cgaatttctg ggcgataacg
      841 ataaatttaa caaagaagtg atgtatgcgt atgtggatca gcatgatttt agcggcaaag
      901 attttgtgag cgcgctgcgc atgtttctgg aaggctttcg cctgccgggc gaagcgcaga
      961 aaattgatcg cctgatggaa aaatttgcgg cgcgctatct ggaatgcaac cagggccaga
     1021 ccctgtttgc gagcgcggat accgcgtatg tgctggcgta tagcattatt atgctgacca
     1081 ccgatctgca tagcccgcag gtgaaaaaca aaatgaccaa agaacagtat attaaaatga
     1141 accgcggcat taacgatagc aaagatctgc cggaagaata tctgagcgcg atttataacg
     1201 aaattgcggg caaaaaaatt agcatgaaat aataaggatc catgggtacc ctcgagtgaa
     1261 gacaagctta ggtatttatt cggcgcaaag tgcgtcgggt gatgctgcca acttagtcga
     1321 gcaccaccac caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt
     1381 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     1441 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
     1501 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     1561 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     1621 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     1681 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
     1741 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
     1801 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
     1861 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
     1921 aacaaaatat taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc
     1981 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag
     2041 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     2101 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     2161 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     2221 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     2281 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     2341 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     2401 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     2461 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     2521 tctaatacct ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca
     2581 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     2641 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     2701 tctggcgcat cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta
     2761 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta
     2821 gagcaagacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     2881 gcagacagtt ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     2941 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     3001 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     3061 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     3121 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     3181 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     3241 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     3301 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     3361 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     3421 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     3481 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     3541 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     3601 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     3661 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     3721 gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg
     3781 cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta
     3841 agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc
     3901 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     3961 ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg
     4021 cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct
     4081 gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa
     4141 agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg
     4201 gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg
     4261 ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat
     4321 ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag
     4381 atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg
     4441 tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc
     4501 atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta
     4561 tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg
     4621 acaggagcac gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct
     4681 cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc
     4741 cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga
     4801 aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca
     4861 taagtgcggc gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg
     4921 ctctcaaggg catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg
     4981 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     5041 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     5101 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     5161 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc
     5221 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     5281 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     5341 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     5401 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     5461 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     5521 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     5581 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     5641 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     5701 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     5761 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     5821 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     5881 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     5941 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     6001 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     6061 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     6121 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc
     6181 ctgcattagg aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa
     6241 tggtgcatg
//
