LOCUS       sequence-470399-        6114 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of K1355A/K1435A double-mutant GST-tagged Spt6 
            tSH2 domain (1223-1453).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6114)
  TITLE     Hill lab spt6-tsh2 plasmids
REFERENCE   2  (bases 1 to 6114)
  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: CPH3737_GST-TEV-Spt6p-tSH2-1223-1453
FEATURES             Location/Qualifiers
     source          1..6114
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     25..44
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        25..43
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             75..97
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             105..758
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     protein_bind    792..816
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             837..857
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     protein_bind    complement(1576..1600)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(1664..1682)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1678..1725
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2091..2096,2097..2113,2114..2119))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2146..2164)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2232..2336
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2337..2405,2406..3197)
                     /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(2555..2574)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3368..3956
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3857..3876
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4110..4127
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4142..4281
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4207..4226)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4367..4389
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4383..4574)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     6036..6055
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 atcgagatct cgatcccgcg aaattaatac gactcactat agggagacca caacggtttc
       61 cctctagaaa taattttgtt taactttaag aaggagatat acatatgtcc cctatactag
      121 gttattggaa aattaagggc cttgtgcaac ccactcgact tcttttggaa tatcttgaag
      181 aaaaatatga agagcatttg tatgagcgcg atgaaggtga taaatggcga aacaaaaagt
      241 ttgaattggg tttggagttt cccaatcttc cttattatat tgatggtgat gttaaattaa
      301 cacagtctat ggccatcata cgttatatag ctgacaagca caacatgttg ggtggttgtc
      361 caaaagagcg tgcagagatt tcaatgcttg aaggagcggt tttggatatt agatacggtg
      421 tttcgagaat tgcatatagt aaagactttg aaactctcaa agttgatttt cttagcaagc
      481 tacctgaaat gctgaaaatg ttcgaagatc gtttatgtca taaaacatat ttaaatggtg
      541 atcatgtaac ccatcctgac ttcatgttgt atgacgctct tgatgttgtt ttatacatgg
      601 acccaatgtg cctggatgcg ttcccaaaat tagtttgttt taaaaaacgt attgaagcta
      661 tcccacaaat tgataagtac ttgaaatcca gcaagtatat agcatggcct ttgcagggct
      721 ggcaagccac gtttggtggt ggcgaccatc ctccaaaatc ggatctggtt ccgcgtccat
      781 ggtcgaatca aacaagtttg tacaaaaaag caggctccgc ggccgccgta ctcgaggaaa
      841 acctgtattt tcagggctcc ttcaccagca tttgggactt gaaacaagaa ctggaagatg
      901 ccgaagagga gaggaaattg atgatggcag aagcccgtgc aaagagaaca catcgtgtta
      961 tcaatcatcc ttactatttc cctttcaacg gcagacaggc tgaggattac ttaaggagta
     1021 aagaacgtgg tgaattcgtg atcagacagt ctagccgagg tgatgaccac ttggttatca
     1081 cctggaaatt ggataaggat ttgtttcaac atattgatat ccaagaatta gaaaaagaaa
     1141 atcctttggc tttaggtaaa gtcttgattg tcgacaatca gaaatacaat gatttagacc
     1201 agatcattgt agaatatctt caaaacaagg taaggctctt gaatgaaatg acatctagtg
     1261 aagcattcaa aagcggtact aagaaagatg tggtcaagtt tattgaagac tactctagag
     1321 tgaatccaaa taagtctatg tactatttca gtttgaacca cgataaccct ggttggtttt
     1381 acttgatgtt caagattaac gcaaatagca aattatacac atggaatgtg aaattaacga
     1441 acactggtta tttcctggta aactacaatt atccaagtgt tatccagctt tgtaatggtt
     1501 ttgcgacgct tctaaaatct aacagtagta agaatagaat gaacaactac cgttagtaga
     1561 agggtgggcg cgccgaccca gctttcttgt acaaagtggt ttgattcgac ccgggatccg
     1621 gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta
     1681 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     1741 atatccggat atccacagga cgggtgtggt cgccatgatc gcgtagtcga tagtggctcc
     1801 aagtagcgaa gcgagcagga ctgggcggcg gccaaagcgg tcggacagtg ctccgagaac
     1861 gggtgcgcat agaaattgca tcaacgcata tagcgctagc agcacgccat agtgactggc
     1921 gatgctgtcg gaatggacga tatcccgcaa gaggcccggc agtaccggca taaccaagcc
     1981 tatgcctaca gcatccaggg tgacggtgcc gaggatgacg atgagcgcat tgttagattt
     2041 catacacggt gcctgactgc gttagcaatt taactgtgat aaactaccgc attaaagctt
     2101 atcgatgata agctgtcaaa catgagaatt cttgaagacg aaagggcctc gtgatacgcc
     2161 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     2221 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2281 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     2341 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     2401 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     2461 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     2521 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg
     2581 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     2641 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     2701 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     2761 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     2821 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     2881 cagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     2941 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     3001 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     3061 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     3121 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     3181 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     3241 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     3301 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3361 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3421 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3481 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     3541 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3601 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3661 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3721 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3781 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3841 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3901 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3961 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4021 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     4081 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     4141 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     4201 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     4261 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4321 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4381 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     4441 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     4501 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     4561 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     4621 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     4681 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     4741 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     4801 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     4861 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     4921 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     4981 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     5041 ggccaggacc caacgctgcc cgagatgcgc cgcgtgcggc tgctggagat ggcggacgcg
     5101 atggatatgt tctgccaagg gttggtttgc gcattcacag ttctccgcaa gaattgattg
     5161 gctccaattc ttggagtggt gaatccgtta gcgaggtgcc gccggcttcc attcaggtcg
     5221 aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg tatagggcgg
     5281 cgcctacaat ccatgccaac ccgttccatg tgctcgccga ggcggcataa atcgccgtga
     5341 cgatcagcgg tccaatgatc gaagttaggc tggtaagagc cgcgagcgat ccttgaagct
     5401 gtccctgatg gtcgtcatct acctgcctgg acagcatggc ctgcaacgcg ggcatcccga
     5461 tgccgccgga agcgagaaga atcataatgg ggaaggccat ccagcctcgc gtcgcgaacg
     5521 ccagcaagac gtagcccagc gcgtcggccg ccatgccggc gataatggcc tgcttctcgc
     5581 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     5641 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     5701 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     5761 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     5821 tcaagggcat cggtcgatcg acgctctccc ttatgcgact cctgcattag gaagcagccc
     5881 agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg
     5941 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     6001 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     6061 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gagg
//
