LOCUS       sequence-432995-        7284 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of 10xHis-SUMO (Smt3) fusion to HRV-3C 
            cleavable Halotagged AP3D1 adaptin appendage (920-1153) for pulldown
            assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7284)
  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 7284)
  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: MZB131
FEATURES             Location/Qualifiers
     source          1..7284
                     /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             633..1523
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     CDS             1539..1562
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             2357..2374
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2427..2445)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2441..2488
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2525..2980
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2612..2631)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2822..2843
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3072..3887)
                     /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(3072..3887)
                     /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     3795..3814
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3879..3898
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      4009..4597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4498..4517
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4751..4768
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4783..4922
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4848..4867)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5008..5030
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5024..5215)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6024..7106)
                     /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     7068..7087
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7107..7184)
                     /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 gcatggcaga aatcggtact ggctttccat
      661 tcgaccccca ttatgtggaa gtcctgggcg agcgcatgca ctacgtcgat gttggtccgc
      721 gcgatggcac ccctgtgctg ttcctgcacg gtaacccgac ctcctcctac gtgtggcgca
      781 acatcatccc gcatgttgca ccgacccatc gctgcattgc tccagacctg atcggtatgg
      841 gcaaatccga caaaccagac ctgggttatt tcttcgacga ccacgtccgc ttcatggatg
      901 ccttcatcga agccctgggt ctggaagagg tcgtcctggt cattcacgac tggggctccg
      961 ctctgggttt ccactgggcc aagcgcaatc cagagcgcgt caaaggtatt gcatttatgg
     1021 agttcatccg ccctatcccg acctgggacg aatggccaga atttgcccgc gagaccttcc
     1081 aggccttccg caccaccgac gtcggccgca agctgatcat cgatcagaac gtttttatcg
     1141 agggtacgct gccgatgggt gtcgtccgcc cgctgactga agtcgagatg gaccattacc
     1201 gcgagccgtt cctgaatcct gttgaccgcg agccactgtg gcgcttccca aacgagctgc
     1261 caatcgccgg tgagccagcg aacatcgtcg cgctggtcga agaatacatg gactggctgc
     1321 accagtcccc tgtcccgaag ctgctgttct ggggcacccc aggcgttctg atcccaccgg
     1381 ccgaagccgc tcgcctggcc aaaagcctgc ctaactgcaa ggctgtggac atcggcccgg
     1441 gtctgaatct gctgcaagaa gacaacccgg acctgatcgg cagcgagatc gcgcgctggc
     1501 tgtcgacgct cgagatttcc ggcgagccaa ccactggcct ggaagtgctg tttcagggcc
     1561 cgtcaagcta ctcattgctt gctgaaaaca gctacgtaaa gatgacttgt gacatccggg
     1621 gctccttgca agaggattcg caggtcaccg tggcaatcgt attggagaac cgctcgtcat
     1681 cgattttgaa aggtatggaa ttgagtgttt tagactctct gaacgcgcgc atggcacgtc
     1741 cccagggcag ctctgtacat gatggtgtgc ccgtcccatt ccaactgccc cccggagtat
     1801 cgaatgaggc ccaatacgta tttacgattc agtcaatcgt aatggcgcaa aagttgaaag
     1861 gaacattgtc ttttatagca aaaaatgacg agggagcgac ccatgagaaa ttggatttcc
     1921 gtttgcattt ctcttgctca tcctatctga ttacgacccc ttgttactct gacgcctttg
     1981 cgaagctgtt ggagagtgga gatttgagta tgagcagtat taaagtcgat gggatacgga
     2041 tgtcattcca gaacctttta gccaagatct gcttccacca tcatttttcc gttgtggagc
     2101 gtgttgactc ctgcgcttca atgtatagtc gttcaataca aggacatcac gtatgtttgc
     2161 tggtgaaaaa aggtgaaaat tctgtttccg tagatggaaa atgttctgac tctacactgt
     2221 taagtaattt attagaagaa atgaaggcta ctttggcaaa atgctaataa ggatccatgg
     2281 gtaccctcga gtgaagacaa gcttaggtat ttattcggcg caaagtgcgt cgggtgatgc
     2341 tgccaactta gtcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg
     2401 aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc
     2461 ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa
     2521 tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2581 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2641 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2701 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     2761 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     2821 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     2881 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     2941 tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa
     3001 atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca
     3061 tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca
     3121 ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg
     3181 aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca
     3241 tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca
     3301 tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt
     3361 tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc
     3421 attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa
     3481 acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct
     3541 gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt
     3601 aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc
     3661 gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca
     3721 tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct
     3781 gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa
     3841 tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta
     3901 ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt
     3961 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4021 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4081 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4141 gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4201 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4261 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4321 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4381 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4441 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4501 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4561 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4621 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     4681 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     4741 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct
     4801 ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct
     4861 gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg
     4921 cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     4981 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5041 catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt
     5101 cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg
     5161 tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg
     5221 cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga
     5281 tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta
     5341 aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc
     5401 gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga
     5461 tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga
     5521 aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc
     5581 acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta
     5641 gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga
     5701 taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga
     5761 gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa
     5821 agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga
     5881 taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc
     5941 tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct
     6001 aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6061 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg
     6121 gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc
     6181 tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg
     6241 atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc
     6301 gagatatccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc
     6361 atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg
     6421 gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt
     6481 tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt
     6541 aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc
     6601 agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca
     6661 tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca
     6721 tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc
     6781 gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt
     6841 tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg
     6901 gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg
     6961 ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg
     7021 tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg
     7081 acatcgtata acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc
     7141 tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc
     7201 tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac
     7261 cgccgccgca aggaatggtg catg
//
