LOCUS       sequence-451122-        5607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of HIS-MBP-TEV tagged Tom1p HECT domain 
            (2850-3268).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5607)
  TITLE     Hill lab spt6-tsh2 plasmids
REFERENCE   2  (bases 1 to 5607)
  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"
COMMENT     Sequence Label: CPH3949_HIS-MBP-TEV-Tom1p-HECT-2850-3268
FEATURES             Location/Qualifiers
     source          1..5607
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(22..40)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        108..212
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(213..281,282..1073)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(431..450)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1244..1832
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1733..1752
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1986..2003
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2018..2157
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2083..2102)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2243..2265
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2259..2450)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     2959..2978
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2959..2977
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             3009..3031
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3042..3071
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
     CDS             3072..4169
                     /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     4143..4166
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             4182..4202
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     RBS             5381..5389
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(5523..5541)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      5537..5584
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
ORIGIN
        1 ataattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
       61 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
      121 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
      181 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
      241 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
      301 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
      361 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
      421 ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa gagcaactcg
      481 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
      541 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
      601 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
      661 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
      721 ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca acgttgcgca
      781 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
      841 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
      901 ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag
      961 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     1021 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     1081 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     1141 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     1201 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     1261 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     1321 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     1381 caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     1441 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     1501 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     1561 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     1621 acctacagcg tgagctatga gaaagcgcca cgcttctcga agggagaaag gcggacaggt
     1681 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     1741 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     1801 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     1861 tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg
     1921 tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg
     1981 agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat tttctcctta
     2041 cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct gctctgatgc
     2101 cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat ggctgcgccc
     2161 cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct
     2221 tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc accgtcatca
     2281 ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag
     2341 atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt taatgtctgg
     2401 cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac tgatgcctcc
     2461 gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga gaggatgctc
     2521 acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga gggtaaacaa
     2581 ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg ccagcgcttc
     2641 gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat gcagatccgg
     2701 aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac acggaaaccg
     2761 aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt
     2821 cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca gcctagccgg
     2881 gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac gctgcccgag
     2941 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta
     3001 gaaataattt tgtttaactt taagaaggag atatacatat gcaccaccac caccaccatc
     3061 atcatcatca taaaactgaa gaaggtaaac tggtaatctg gattaacggc gataaaggct
     3121 ataacggtct cgctgaagtc ggtaagaaat tcgagaaaga taccggaatt aaagtcaccg
     3181 ttgagcatcc ggataaactg gaagagaaat tcccacaggt tgcggcaact ggcgatggcc
     3241 ctgacattat cttctgggca cacgaccgct ttggtggcta cgctcaatct ggcctgttgg
     3301 ctgaaatcac cccggacaaa gcgttccagg acaagctgta tccgtttacc tgggatgccg
     3361 tacgttacaa cggcaagctg attgcttacc cgatcgctgt tgaagcgtta tcgctgattt
     3421 ataacaaaga tctgctgccg aacccgccaa aaacctggga agagatcccg gcgctggata
     3481 aagaactgaa agcgaaaggt aagagcgcgc tgatgttcaa cctgcaagaa ccgtacttca
     3541 cctggccgct gattgctgct gacgggggtt atgcgttcaa gtatgaaaac ggcaagtacg
     3601 acattaaaga cgtgggcgtg gataacgctg gcgcgaaagc gggtctgacc ttcctggttg
     3661 acctgattaa aaacaaacac atgaatgcag acaccgatta ctccatcgca gaagctgcct
     3721 ttaataaagg cgaaacagcg atgaccatca acggcccgtg ggcatggtcc aacatcgaca
     3781 ccagcaaagt gaattatggt gtaacggtac tgccgacctt caagggtcaa ccatccaaac
     3841 cgttcgttgg cgtgctgagc gcaggtatta acgccgccag tccgaacaaa gagctggcaa
     3901 aagagttcct cgaaaactat ctgctgactg atgaaggtct ggaagcggtt aataaagaca
     3961 aaccgctggg tgccgtagcg ctgaagtctt acgaggaaga gttggcgaaa gatccacgta
     4021 ttgccgccac tatggaaaac gcccagaaag gtgaaatcat gccgaacatc ccgcagatgt
     4081 ccgctttctg gtatgccgtg cgtactgcgg tgatcaacgc cgccagcggt cgtcagactg
     4141 tcgatgaagc cctgaaagac gcgcagacta attcgagctc cgagaatctt tattttcagg
     4201 gatccaactt atttttccca tttacagacg cacacaagaa gttgttgaat caaatgattc
     4261 gttcaaatcc aaaactgatg agtggtcctt tcgcattatt ggtcaagaat ccaaaagttt
     4321 tggattttga caacaaaaga tatttcttca atgccaaatt gaagtctgat aatcaagagc
     4381 gtcctaaatt acctattacc gttcgccgtg agcaggtttt cttggactct tatcgagcct
     4441 tatttttcaa gacaaacgat gaaatcaaga attctaaatt agagataaca ttcaaaggag
     4501 aaagtggtgt cgatgccggt ggggtaacca gagaatggta ccaagtacta tcgagacaga
     4561 tgttcaaccc tgattatgct ctttttcttc ctgtaccatc ggataaaact actttccatc
     4621 ctaatcggac atcaggtatc aaccctgagc acctttcttt ctttaaattt atcgggatga
     4681 ttatagggaa agcaattaga gaccaatgct ttttggattg ccattttagt cgtgaagtat
     4741 acaagaatat tttgggtagg cctgtatcat tgaaggatat ggagtctctt gatccagatt
     4801 attataaatc attggtgtgg attcttgaaa acgatatcac agatattata gaggaaactt
     4861 tttctgttga aactgatgat tatggtgaac ataaagttat caatttgatt gaggggggca
     4921 aggatataat agttacagag gccaataaac aggattatgt gaaaaaggtt gtcgaatata
     4981 aacttcagac atctgttaag gaacaaatgg ataacttttt agtgggtttc tatgccctta
     5041 tatccaaaga tctcattaca atatttgatg agcaggaatt ggagttatta atcagcggtt
     5101 tgcctgatat tgatgtcgac gactggaaaa acaatactac ttatgttaat tacaccgcaa
     5161 cctgtaagga agtgagttat ttttggaggg cagttagatc cttcgacgct gaagaacgtg
     5221 ccaagttgtt gcagtttgtt accggtacta gtaaagttcc tctaaatggg tttaaggaac
     5281 taagcggtgt caacggtgtt tgtaaatttt ccatccatcg tgattttggt tcatcagaaa
     5341 gactaccatc atcacatacc tgtttcaatc aactgaattt acctccttat gaatcatatg
     5401 agacattgcg tggttcacta ttattggcaa tcaatgaagg gcatgaaggg tttggtcttg
     5461 cctagctcga gcagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc
     5521 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     5581 tttgctgaaa ggaggaacta tatccgg
//
