LOCUS       sequence-455977-        7122 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of 10xHis-SUMO (Smt3) fusion to HRV-3C 
            cleavable Halotagged Arf (T31N) for pulldown assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7122)
  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 7122)
  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: MZB224
FEATURES             Location/Qualifiers
     source          1..7122
                     /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             2195..2212
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2265..2283)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2279..2326
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2363..2818
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2450..2469)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2660..2681
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2910..3725)
                     /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(2910..3725)
                     /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     3633..3652
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3717..3736
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3847..4435
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4336..4355
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4589..4606
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4621..4760
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4686..4705)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4846..4868
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4862..5053)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5862..6944)
                     /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     6906..6925
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6945..7022)
                     /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 cgggtaatat attcgccaac ttgttcaagg gcttgttcgg taaaaaagag atgcgcattt
     1621 tgatggtcgg tctggacgct gccggcaaaa cgaacatcct gtataagctg aaattagggg
     1681 aaatcgtgac taccattcct acaatcggct ttaacgtaga aacagttgaa tataagaata
     1741 tcagttttac tgtatgggat gtagggggtc aggataagat cagaccttta tggagacact
     1801 atttccaaaa tacgcaggga ctgatctttg tagtagactc taatgaccgt gagcgggtga
     1861 atgaagcccg cgaagaactt atgcgcatgc ttgcagaaga cgagctgaga gatgcggtcc
     1921 tgttggtgtt cgccaataaa caagatttac cgaacgccat gaacgctgca gaaataactg
     1981 acaaattggg acttcactcc ctgcggcata gaaactggta cattcaggca acgtgcgcaa
     2041 cgagcggcga cgggctgtac gagggtttgg actggcttag caaccaactg cggaatcaaa
     2101 aataataagg atccatgggt accctcgagt gaagacaagc ttaggtattt attcggcgca
     2161 aagtgcgtcg ggtgatgctg ccaacttagt cgagcaccac caccaccacc actgagatcc
     2221 ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg agcaataact
     2281 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     2341 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     2401 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     2461 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     2521 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     2581 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     2641 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     2701 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     2761 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttag
     2821 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2881 caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat caaatgaaac
     2941 tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg tttctgtaat
     3001 gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta tcggtctgcg
     3061 attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa aataaggtta
     3121 tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa aagtttatgc
     3181 atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa atcactcgca
     3241 tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac gcgatcgctg
     3301 ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac tgccagcgca
     3361 tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc tgttttcccg
     3421 gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg cttgatggtc
     3481 ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt aacatcattg
     3541 gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt cccatacaat
     3601 cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata cccatataaa
     3661 tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg ttgaatatgg
     3721 ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt tcatgaccaa
     3781 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     3841 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     3901 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     3961 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     4021 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     4081 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     4141 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     4201 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     4261 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     4321 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     4381 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     4441 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     4501 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     4561 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     4621 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     4681 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     4741 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     4801 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     4861 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     4921 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     4981 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     5041 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     5101 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     5161 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     5221 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     5281 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     5341 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     5401 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     5461 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
     5521 gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg gcgggaccag
     5581 tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca
     5641 tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct gccggcacct
     5701 gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata gtcatgcccc
     5761 gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt cgagatcccg
     5821 gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc gctttccagt
     5881 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5941 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
     6001 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
     6061 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
     6121 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
     6181 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
     6241 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
     6301 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
     6361 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
     6421 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
     6481 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
     6541 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
     6601 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
     6661 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
     6721 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
     6781 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
     6841 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
     6901 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     6961 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     7021 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     7081 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tg
//
