LOCUS       sequence-181072-        6378 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of residues 1247-1451 from S. cerevisiae Spt6 
            with a K1435A 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: CPH3495 (pET151_Spt6 1247-1451 K1435A)
FEATURES             Location/Qualifiers
     source          1..6378
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     116..135
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     219..238
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        219..237
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    238..262
                     /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             277..299
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             310..327
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             328..369
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             370..390
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1089..1107)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1103..1150
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1388..1406)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1474..1578
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1579..1647,1648..2439)
                     /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(1797..1816)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2610..3198
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3099..3118
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3352..3369
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3384..3523
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3449..3468)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3609..3631
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3625..3816)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5128..6210)
                     /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     6172..6191
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6211..6288)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
       61 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
      121 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
      181 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
      241 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
      301 atacatatgc atcatcacca tcaccatggt aagcctatcc ctaaccctct cctcggtctc
      361 gattctacgg aaaacctgta ttttcaggga attgatccct tcaccgcaaa gagaacacat
      421 cgtgttatca atcatcctta ctatttccct ttcaacggca gacaggctga ggattactta
      481 aggagtaaag aacgtggtga attcgtgatc agacagtcta gccgaggtga tgaccacttg
      541 gttatcacct ggaaattgga taaggatttg tttcaacata ttgatatcca agaattagaa
      601 aaagaaaatc ctttggcttt aggtaaagtc ttgattgtcg acaatcagaa atacaatgat
      661 ttagaccaga tcattgtaga atatcttcaa aacaaggtaa ggctcttgaa tgaaatgaca
      721 tctagtgaaa aattcaaaag cggtactaag aaagatgtgg tcaagtttat tgaagactac
      781 tctagagtga atccaaataa gtctgtgtac tatttcagtt tgaaccacga taaccctggt
      841 tggttttact tgatgttcaa gattaacgca aatagcaaat tatacacatg gaatgtgaaa
      901 ttaacgaaca ctggttattt cctggtaaac tacaattatc caagtgttat ccagctttgt
      961 aatggttttg cgacgcttct aaaatctaac agtagtaaga atagaatgaa caactaccgt
     1021 tagtagaagg gcgagctcag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     1081 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     1141 gggttttttg ctgaaaggag gaactatatc cggatatccc gcaagaggcc cggcagtacc
     1201 ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat gacgatgagc
     1261 gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg tgataaacta
     1321 ccgcattaaa gctagcttat cgatgataag ctgtcaaaca tgagaattaa ttcttgaaga
     1381 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     1441 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1501 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     1561 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     1621 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     1681 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     1741 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     1801 tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac
     1861 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     1921 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     1981 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     2041 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     2101 gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc
     2161 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     2221 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     2281 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     2341 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     2401 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     2461 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     2521 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2581 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2641 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2701 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     2761 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2821 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2881 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2941 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3001 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3061 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3121 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3181 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3241 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3301 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3361 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     3421 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     3481 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     3541 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     3601 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     3661 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     3721 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     3781 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     3841 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     3901 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     3961 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     4021 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     4081 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     4141 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     4201 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     4261 gagcacgatc atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg
     4321 gctgctggag atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac
     4381 agttctccgc aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg
     4441 ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg
     4501 aggcagacaa ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc
     4561 gaggcggcat aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga
     4621 gccgcgagcg atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg
     4681 gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc
     4741 atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg
     4801 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga
     4861 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag
     4921 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc
     4981 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag
     5041 gagctgactg ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg
     5101 agctaactta cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     5161 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     5221 cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg
     5281 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     5341 tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac
     5401 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     5461 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     5521 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     5581 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     5641 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     5701 gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga
     5761 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     5821 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     5881 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     5941 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     6001 actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     6061 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     6121 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     6181 tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg
     6241 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac
     6301 gctctccctt atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga
     6361 gcaccgccgc cgcaagga
//
