LOCUS       sequence-181071-        6378 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of residues 1247-1451 from S. cerevisiae Spt6 
            with an R1282H mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6378)
  AUTHORS   Sdano MA, Fulcher JM, Palani S, Chandrasekharan MB, Parnell TJ, 
            Whitby FG, Formosa T, Hill CP
  TITLE     A novel SH2 recognition mechanism recruits Spt6 to the doubly 
            phosphorylated RNA polymerase II linker at sites of transcription.
  JOURNAL   Elife. 2017 Aug 16;6. pii: 28723. doi: 10.7554/eLife.28723.
  PUBMED    28826505
REFERENCE   2  (bases 1 to 6378)
  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: "Elife."; 
            date: "2017-08-16"; volume: "6. pii: 28723. doi"; pages: " 
            10.7554/eLife.28723"
COMMENT     Sequence Label: CPH3567 (pET151_Spt6 1247-1451 R1282H)
FEATURES             Location/Qualifiers
     source          1..6378
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     100..119
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     203..222
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        203..221
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    222..246
                     /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             261..283
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             294..311
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             312..353
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             354..374
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1073..1091)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1087..1134
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1372..1390)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1458..1562
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1563..1631,1632..2423)
                     /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(1781..1800)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2594..3182
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3083..3102
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3336..3353
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3368..3507
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3433..3452)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3593..3615
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3609..3800)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5112..6194)
                     /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     6156..6175
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6195..6272)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc
       61 gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc
      121 gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat
      181 cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac
      241 aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacat atgcatcatc
      301 accatcacca tggtaagcct atccctaacc ctctcctcgg tctcgattct acggaaaacc
      361 tgtattttca gggaattgat cccttcaccg caaagagaac acatcgtgtt atcaatcatc
      421 cttactattt ccctttcaac ggcagacagg ctgaggatta cttaaggagt aaagaacgtg
      481 gtgaattcgt gatccatcag tctagccgag gtgatgacca cttggttatc acctggaaat
      541 tggataagga tttgtttcaa catattgata tccaagaatt agaaaaagaa aatcctttgg
      601 ctttaggtaa agtcttgatt gtcgacaatc agaaatacaa tgatttagac cagatcattg
      661 tagaatatct tcaaaacaag gtaaggctct tgaatgaaat gacatctagt gaaaaattca
      721 aaagcggtac taagaaagat gtggtcaagt ttattgaaga ctactctaga gtgaatccaa
      781 ataagtctgt gtactatttc agtttgaacc acgataaccc tggttggttt tacttgatgt
      841 tcaagattaa cgcaaatagc aaattataca catggaatgt gaaattaacg aacactggtt
      901 atttcctggt aaactacaat tatccaagtg ttatccagct ttgtaatggt tttaagacgc
      961 ttctaaaatc taacagtagt aagaatagaa tgaacaacta ccgttagtag aagggcgagc
     1021 tcagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag
     1081 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     1141 ggaggaacta tatccggata tcccgcaaga ggcccggcag taccggcata accaagccta
     1201 tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca
     1261 tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat taaagctagc
     1321 ttatcgatga taagctgtca aacatgagaa ttaattcttg aagacgaaag ggcctcgtga
     1381 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     1441 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     1501 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     1561 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     1621 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     1681 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     1741 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     1801 cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     1861 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     1921 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     1981 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     2041 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     2101 tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     2161 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     2221 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     2281 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     2341 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     2401 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     2461 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     2521 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2581 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2641 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2701 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     2761 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2821 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2881 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2941 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     3001 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     3061 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     3121 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3181 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     3241 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3301 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3361 aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcaatg
     3421 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta
     3481 tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc
     3541 tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc
     3601 tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc
     3661 tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg
     3721 ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg
     3781 gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta
     3841 atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacatgcc
     3901 cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga
     3961 aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt tccacagggt
     4021 agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc tgacttccgc
     4081 gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc tcaggtcgca
     4141 gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc attctgctaa
     4201 ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac gatcatgcgc
     4261 acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct ggagatggcg
     4321 gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct ccgcaagaat
     4381 tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg gcttccattc
     4441 aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag acaaggtata
     4501 gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg gcataaatcg
     4561 ccgtgacgat cagcggtcca atgatcgaag ttaggctggt aagagccgcg agcgatcctt
     4621 gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc aacgcgggca
     4681 tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag cctcgcgtcg
     4741 cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata atggcctgct
     4801 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     4861 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     4921 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     4981 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga
     5041 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     5101 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5161 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     5221 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     5281 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     5341 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     5401 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     5461 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     5521 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     5581 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5641 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5701 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5761 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag
     5821 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5881 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5941 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     6001 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     6061 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     6121 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     6181 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     6241 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     6301 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     6361 gaatggtgca tgcaagga
//
