LOCUS       sequence-176493-        6381 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of residues 1247-1451 from S. cerevisiae Spt6 
            with K1435A and K1355A mutations.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6381)
  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 6381)
  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: CPH3506 (pET151_Spt6 1247-1451 K1355A,K1435A)
FEATURES             Location/Qualifiers
     source          1..6381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             300..317
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             318..359
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             360..380
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1079..1097)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1093..1140
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1378..1396)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1464..1568
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1569..1637,1638..2429)
                     /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(1787..1806)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2600..3188
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3089..3108
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3342..3359
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3374..3516
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3442..3461)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3602..3624
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3618..3809)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5121..6203)
                     /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     6165..6184
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6204..6281)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc
      301 atcatcacca tcaccatggt aagcctatcc ctaaccctct cctcggtctc gattctacgg
      361 aaaacctgta ttttcaggga attgatccct tcaccgcaaa gagaacacat cgtgttatca
      421 atcatcctta ctatttccct ttcaacggca gacaggctga ggattactta aggagtaaag
      481 aacgtggtga attcgtgatc agacagtcta gccgaggtga tgaccacttg gttatcacct
      541 ggaaattgga taaggatttg tttcaacata ttgatatcca agaattagaa aaagaaaatc
      601 ctttggcttt aggtaaagtc ttgattgtcg acaatcagaa atacaatgat ttagaccaga
      661 tcattgtaga atatcttcaa aacaaggtaa ggctcttgaa tgaaatgaca tctagtgaag
      721 cgttcaaaag cggtactaag aaagatgtgg tcaagtttat tgaagactac tctagagtga
      781 atccaaataa gtctgtgtac tatttcagtt tgaaccacga taaccctggt tggttttact
      841 tgatgttcaa gattaacgca aatagcaaat tatacacatg gaatgtgaaa ttaacgaaca
      901 ctggttattt cctggtaaac tacaattatc caagtgttat ccagctttgt aatggttttg
      961 cgacgcttct aaaatctaac agtagtaaga atagaatgaa caactaccgt tagtagaagg
     1021 gcgagctcag atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc
     1081 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
     1141 ctgaaaggag gaactatatc cggatatccc gcaagaggcc cggcagtacc ggcataacca
     1201 agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc gcattgttag
     1261 atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta ccgcattaaa
     1321 gctagcttat cgatgataag ctgtcaaaca tgagaattaa ttcttgaaga cgaaagggcc
     1381 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     1441 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     1501 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     1561 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     1621 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     1681 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     1741 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     1801 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     1861 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     1921 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     1981 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     2041 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     2101 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     2161 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     2221 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     2281 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     2341 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     2401 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     2461 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     2521 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2581 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2641 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2701 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     2761 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2821 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2881 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2941 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3001 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3061 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     3121 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3181 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3241 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     3301 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3361 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3421 gcatatatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3481 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3541 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3601 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     3661 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     3721 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     3781 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     3841 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     3901 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     3961 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     4021 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     4081 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     4141 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4201 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     4261 atcatgcgca cccgtggcca ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg
     4321 gagatggcgg acgcgatgga tatgttctgc caagggttgg tttgcgcatt cacagttctc
     4381 cgcaagaatt gattggctcc aattcttgga gtggtgaatc cgttagcgag gtgccgccgg
     4441 cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga
     4501 caaggtatag ggcggcgcct acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg
     4561 cataaatcgc cgtgacgatc agcggtccag tgatcgaagt taggctggta agagccgcga
     4621 gcgatccttg aagctgtccc tgatggtcgt catctacctg cctggacagc atggcctgca
     4681 acgcgggcat cccgatgccg ccggaagcga gaagaatcat aatggggaag gccatccagc
     4741 ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa
     4801 tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg
     4861 cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc
     4921 ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt tgcatgataa
     4981 agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg aaggagctga
     5041 ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga gtgagctaac
     5101 ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     5161 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgccagggtg
     5221 gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc ctggccctga
     5281 gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg
     5341 gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc cactaccgag
     5401 atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc cagcgccatc
     5461 tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat ttgcatggtt
     5521 tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg ctgaatttga
     5581 ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac agaacttaat
     5641 gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc cacgcccagt
     5701 cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc agagacatca
     5761 agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc ctggtcatcc
     5821 agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg caccgccgct
     5881 ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc acccagttga
     5941 tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc cagactggag
     6001 gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac gcggttggga
     6061 atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc agaaacgtgg
     6121 ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata ctctgcgaca
     6181 tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc cgggcgctat
     6241 catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc gacgctctcc
     6301 cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc
     6361 cgccgcaagg aatggtgcat g
//
