LOCUS       sequence-311422-        4826 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For C-terminal his6-TEV-ProtA-tagging in S. pombe using KanMX6 as 
            resistance marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4826)
  AUTHORS   Kilchert C, Wittmann S, Passoni M, Shah S, Granneman S, Vasiljeva L
  TITLE     Regulation of mRNA Levels by Decay-Promoting Introns that Recruit 
            the Exosome Specificity Factor Mmi1.
  JOURNAL   Cell Rep. 2015 Dec 22;13(11):2504-2515. doi: 
            10.1016/j.celrep.2015.11.026. Epub 2015 Dec 6.
  PUBMED    26670050
REFERENCE   2  (bases 1 to 4826)
  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"; doi: 
            "10.1016/j.celrep.2015.11.026"; journalName: "Cell Rep"; date: 
            "2015-12-22- 22"; volume: "13"; issue: "11"; pages: "2504-2515"
COMMENT     Sequence Label: pBS1479-HTP-KanMX6
FEATURES             Location/Qualifiers
     source          1..4826
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     134..154
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        136..154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             181..198
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             226..246
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             286..459
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNDAQAPK"
     CDS             460..633
                     /codon_start=1
                     /product="IgG-binding unit of Staphylococcus aureus protein
                     A"
                     /label=ProtA
                     /translation="VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLL
                     AEAKKLNGAQAPK"
     gene            complement(735..2091)
                     /label=kanMX
                     /note="yeast selectable marker conferring kanamycin 
                     resistance (Wach et al., 1994)"
     terminator      complement(735..932)
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     CDS             complement(938..1747)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MGKEKTHVSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1661..1680
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(1748..2091)
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     primer_bind     complement(2146..2165)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2147..2165)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2175..2192)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2175..2191)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2184..2206)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2332..2788)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2470..2491)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2682..2701
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2815..2919
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2920..2988,2989..3780)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3138..3157)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3951..4539
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4440..4459
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4693..4710
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      121 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagg atccatggag
      181 caccatcacc atcaccatga ttatgatatt ccaactactg ctagcgagaa tttgtatttt
      241 cagggtgagc tcaaaaccgc ggctcttgcg caacacgatg aagccgtgga caacaaattc
      301 aacaaagaac aacaaaacgc gttctatgag atcttacatt tacctaactt aaacgaagaa
      361 caacgaaacg ccttcatcca aagtttaaaa gatgacccaa gccaaagcgc taacctttta
      421 gcagaagcta aaaagctaaa tgatgctcag gcgccgaaag tagacaacaa attcaacaaa
      481 gaacaacaaa acgcgttcta tgagatctta catttaccta acttaaacga agaacaacga
      541 aacgccttca tccaaagttt aaaagatgac ccaagccaaa gcgctaacct tttagcagaa
      601 gctaaaaagc taaatggtgc tcaggcgccg aaagtagacg cgaattccgc ggggaagtca
      661 acctgaagct tgatatccat cgatgaattc gagctcgttt aaactggatg gcggcgttag
      721 tatcgaatcg acagcagtat agcgaccagc attcacatac gattgacgca tgatattact
      781 ttctgcgcac ttaacttcgc atctgggcag atgatgtcga ggcgaaaaaa aatataaatc
      841 acgctaacat ttgattaaaa tagaacaact acaatataaa aaaactatac aaatgacaag
      901 ttcttgaaaa caagaatctt tttattgtca gtactgatta gaaaaactca tcgagcatca
      961 aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt
     1021 tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc
     1081 ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa
     1141 taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa
     1201 gcttatgcat ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat
     1261 cactcgcatc aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc
     1321 gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg
     1381 ccagcgcatc aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg
     1441 ttttgccggg gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct
     1501 tgatggtcgg aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa
     1561 catcattggc aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc
     1621 catacaatcg atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc
     1681 catataaatc agcatccatg ttggaattta atcgcggcct cgaaacgtga gtcttttcct
     1741 tacccatggt tgtttatgtt cggatgtgat gtgagaactg tatcctagca agattttaaa
     1801 aggaagtata tgaaagaaga acctcagtgg caaatcctaa ccttttatat ttctctacag
     1861 gggcgcggcg tggggacaat tcaacgcgtc tgtgagggga gcgtttccct gctcgcaggt
     1921 ctgcagcgag gagccgtaat ttttgcttcg cgccgtgcgg ccatcaaaat gtatggatgc
     1981 aaatgattat acatggggat gtatgggcta aatgtacggg cgacagtcac atcatgcccc
     2041 tgagctgcgc acgtcaagac tgtcaaggag ggtattctgg gcctccatgt cgctggccgg
     2101 gtgacccggc ggggacgagg caagctaaac agggtaccca attcgcccta tagtgagtcg
     2161 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     2221 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     2281 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaaattgtaa
     2341 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca tttttttaac
     2401 caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg
     2461 agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa
     2521 gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt
     2581 tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt
     2641 agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga
     2701 gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc
     2761 gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt ttcggggaaa tgtgcgcgga
     2821 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     2881 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     2941 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3001 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3061 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3121 agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag
     3181 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3241 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3301 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3361 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3421 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg
     3481 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     3541 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     3601 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     3661 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     3721 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     3781 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     3841 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     3901 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     3961 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4021 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4081 cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct
     4141 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4201 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4261 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4321 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4381 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4441 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     4501 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     4561 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     4621 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     4681 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg
     4741 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     4801 aaagcgggca gtgagcgcaa cgcaat
//
