LOCUS       sequence-432998-        7122 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of 10xHis-SUMO (Smt3) fusion to HRV-3C 
            cleavable Halotagged Arf (L8K+Q71L) 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: MZB160
FEATURES             Location/Qualifiers
     source          1..7122
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(215..238)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(254..1144)
                     /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             complement(1445..1474)
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     RBS             1488..1510
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1525..1549
                     /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)."
     primer_bind     complement(1549..1568)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1550..1568)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1652..1671)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1877..1954
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1955..3037
                     /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     complement(1974..1993)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3846..4037
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4031..4053)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4139..4278
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4194..4213
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4293..4310)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4464..5052)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4544..4563)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5163..5182)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5174..5989
                     /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             5174..5989
                     /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     complement(5247..5266)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6081..6536)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6218..6239)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6430..6449
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(6573..6620)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     6616..6634
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(6687..6704)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 tatccagacc ccctacatcc catacagtaa aactgatatt cttatattca actgtttcta
       61 cgttaaagcc gattgtagga atggtagtca cgatttcccc taatttcagc ttatacagga
      121 tagtcgtttt gccggcagcg tccagaccga ccatcaaaat gcgcatctct tttttaccga
      181 acaagccctt gaatttgttg gcgaatatat ttcccgggcc ctgaaacagc acttccaggc
      241 cagtggttgg ctcgccggaa atctcgagcg tcgacagcca gcgcgcgatc tcgctgccga
      301 tcaggtccgg gttgtcttct tgcagcagat tcagacccgg gccgatgtcc acagccttgc
      361 agttaggcag gcttttggcc aggcgagcgg cttcggccgg tgggatcaga acgcctgggg
      421 tgccccagaa cagcagcttc gggacagggg actggtgcag ccagtccatg tattcttcga
      481 ccagcgcgac gatgttcgct ggctcaccgg cgattggcag ctcgtttggg aagcgccaca
      541 gtggctcgcg gtcaacagga ttcaggaacg gctcgcggta atggtccatc tcgacttcag
      601 tcagcgggcg gacgacaccc atcggcagcg taccctcgat aaaaacgttc tgatcgatga
      661 tcagcttgcg gccgacgtcg gtggtgcgga aggcctggaa ggtctcgcgg gcaaattctg
      721 gccattcgtc ccaggtcggg atagggcgga tgaactccat aaatgcaata cctttgacgc
      781 gctctggatt gcgcttggcc cagtggaaac ccagagcgga gccccagtcg tgaatgacca
      841 ggacgacctc ttccagaccc agggcttcga tgaaggcatc catgaagcgg acgtggtcgt
      901 cgaagaaata acccaggtct ggtttgtcgg atttgcccat accgatcagg tctggagcaa
      961 tgcagcgatg ggtcggtgca acatgcggga tgatgttgcg ccacacgtag gaggaggtcg
     1021 ggttaccgtg caggaacagc acaggggtgc catcgcgcgg accaacatcg acgtagtgca
     1081 tgcgctcgcc caggacttcc acataatggg ggtcgaatgg aaagccagta ccgatttctg
     1141 ccatgccgcc aatctgttca cgatgcgctt caataatatc gttatcttcc atatccagat
     1201 cttccggggt ctgatccgcc tgaatacgaa tgccatcata cagaaaacgc aggctatcca
     1261 tttctttgcc ctgacgtttc gcaaacgctt ccatcagacg acgcagcggg gtggtttttt
     1321 taattttaaa aaaaatttcg ctgctgccat cgctcacttt caggttaata tgggtttccg
     1381 gtttcacttc cggtttcact tccggtttcg cttcctggtt cacttcgcta tcgctcatgc
     1441 tgccgtgatg gtgatggtga tggtgatggt gatggcccat atgtatatct ccttcttaaa
     1501 gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag
     1561 tcgtattaat ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc
     1621 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
     1681 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
     1741 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg
     1801 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
     1861 gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
     1921 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     1981 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     2041 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     2101 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     2161 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     2221 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     2281 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     2341 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     2401 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     2461 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
     2521 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
     2581 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
     2641 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
     2701 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
     2761 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
     2821 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
     2881 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
     2941 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     3001 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
     3061 agctcactca ttaggcaccg ggatctcgac cgatgccctt gagagccttc aacccagtca
     3121 gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
     3181 tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc gaggaccgct
     3241 ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc ttgcacgccc
     3301 tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag caggccatta
     3361 tcgccggcat ggcggcccca cgggtgcgca tgatcgtgct cctgtcgttg aggacccggc
     3421 taggctggcg gggttgcctt actggttagc agaatgaatc accgatacgc gagcgaacgt
     3481 gaagcgactg ctgctgcaaa acgtctgcga cctgagcaac aacatgaatg gtcttcggtt
     3541 tccgtgtttc gtaaagtctg gaaacgcgga agtcagcgcc ctgcaccatt atgttccgga
     3601 tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta ttaacgaagc
     3661 gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc gccagttgtt
     3721 taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt atcgtgagca
     3781 tcctctctcg tttcatcggt atcattaccc ccatgaacag aaatccccct tacacggagg
     3841 catcagtgac caaacaggaa aaaaccgccc ttaacatggc ccgctttatc agaagccaga
     3901 cattaacgct tctggagaaa ctcaacgagc tggacgcgga tgaacaggca gacatctgtg
     3961 aatcgcttca cgaccacgct gatgagcttt accgcagctg cctcgcgcgt ttcggtgatg
     4021 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     4081 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg
     4141 cagccatgac ccagtcacgt agcgatagcg gagtgtatac tggcttaact atgcggcatc
     4201 agagcagatt gtactgagag tgcaccattg cggtgtgaaa taccgcacag atgcgtaagg
     4261 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     4321 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     4381 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     4441 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     4501 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     4561 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     4621 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     4681 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     4741 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     4801 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     4861 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc
     4921 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     4981 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     5041 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     5101 aactcacgtt aagggatttt ggtcatgaac aataaaactg tctgcttaca taaacagtaa
     5161 tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctctaggc cgcgattaaa
     5221 ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc
     5281 aggtgcgaca atctatcgat tgtatgggaa gcccgatgcg ccagagttgt ttctgaaaca
     5341 tggcaaaggt agcgttgcca atgatgttac agatgagatg gtcagactaa actggctgac
     5401 ggaatttatg cctcttccga ccatcaagca ttttatccgt actcctgatg atgcatggtt
     5461 actcaccact gcgatccccg ggaaaacagc attccaggta ttagaagaat atcctgattc
     5521 aggtgaaaat attgttgatg cgctggcagt gttcctgcgc cggttgcatt cgattcctgt
     5581 ttgtaattgt ccttttaaca gcgatcgcgt atttcgtctc gctcaggcgc aatcacgaat
     5641 gaataacggt ttggttgatg cgagtgattt tgatgacgag cgtaatggct ggcctgttga
     5701 acaagtctgg aaagaaatgc ataaactttt gccattctca ccggattcag tcgtcactca
     5761 tggtgatttc tcacttgata accttatttt tgacgagggg aaattaatag gttgtattga
     5821 tgttggacga gtcggaatcg cagaccgata ccaggatctt gccatcctat ggaactgcct
     5881 cggtgagttt tctccttcat tacagaaacg gctttttcaa aaatatggta ttgataatcc
     5941 tgatatgaat aaattgcagt ttcatttgat gctcgatgag tttttctaag aattaattca
     6001 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6061 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     6121 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     6181 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     6241 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     6301 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     6361 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     6421 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     6481 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca
     6541 ttcgccaatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
     6601 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
     6661 cgggctttgt tagcagccgg atctcagtgg tggtggtggt ggtgctcgac taagttggca
     6721 gcatcacccg acgcactttg cgccgaataa atacctaagc ttgtcttcac tcgagggtac
     6781 ccatggatcc ttattatttt tgattccgca gttggttgct aagccagtcc aaaccctcgt
     6841 acagcccgtc gccgctcgtt gcgcacgttg cctgaatgta ccagtttcta tgccgcaggg
     6901 agtgaagtcc caatttgtca gttatttctg cagcgttcat ggcgttcggt aaatcttgtt
     6961 tattggcgaa caccaacagg accgcatctc tcagctcgtc ttctgcaagc atgcgcataa
     7021 gttcttcgcg ggcttcattc acccgctcac ggtcattaga gtctactaca aagatcagtc
     7081 cctgcgtatt ttggaaatag tgtctccata aaggtctgat ct
//
