LOCUS       sequence-364393-        7057 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of 6xHis C-terminal domain of POLR2A mCherry 
            Fusion Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7057)
  AUTHORS   Asimi V, Sampath Kumar A, Niskanen H, Riemenschneider C, Hetzel S, 
            Naderi J, Fasching N, Popitsch N, Du M, Kretzmer H, Smith ZD, 
            Weigert R, Walther M, Mamde S, Meierhofer D, Wittler L, Buschow R, 
            Timmermann B, Cisse II, Ameres SL, Meissner A, Hnisz D
  TITLE     Hijacking of transcriptional condensates by endogenous retroviruses.
  JOURNAL   Nat Genet. 2022 Aug;54(8):1238-1247. doi: 
            10.1038/s41588-022-01132-w. Epub 2022 Jul 21.
  PUBMED    35864192
REFERENCE   2  (bases 1 to 7057)
  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: 
            "10.1038/s41588-022-01132-w"; journalName: "Nat Genet"; date: 
            "2022-08"; volume: "54"; issue: "8"; pages: "1238-1247"
COMMENT     Sequence Label: pET-45b-POLR2A-CTD-mCherry
FEATURES             Location/Qualifiers
     source          1..7057
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     71..90
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     324..341
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    356..495
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(421..440)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     581..603
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(597..788)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1360..2442)
                     /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     2404..2423
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2443..2520)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2726..2745
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2833..2852
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2833..2851
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2852..2876
                     /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             2891..2913
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2923..2940
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2950..2970
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             4855..4899
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4937..4955)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4951..4998
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      5035..5490
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5122..5141)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5332..5353
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5605..6396,6397..6465))
                     /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     6228..6247
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6466..6558)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6639..7057,1..170)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
       61 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
      121 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
      181 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
      241 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
      301 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga
      361 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag
      421 tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac
      481 tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt
      541 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag
      601 aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg
      661 tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc
      721 agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt
      781 ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg
      841 aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa
      901 cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag
      961 ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat
     1021 cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt
     1081 tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag
     1141 cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa
     1201 ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgctagt catgccccgc
     1261 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     1321 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     1381 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     1441 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     1501 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     1561 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     1621 gtcgtatccc actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg
     1681 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     1741 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     1801 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     1861 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     1921 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     1981 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     2041 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     2101 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     2161 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     2221 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     2281 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     2341 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     2401 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     2461 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc
     2521 cgggatctcg acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt
     2581 tgaggccgtt gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca
     2641 gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga
     2701 agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac
     2761 ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg
     2821 atcccgcgaa attaatacga ctcactatag gggaattgtg agcggataac aattcccctc
     2881 tagaaataat tttgtttaac tttaagaagg agatatacca tgcaccatca ccatcaccat
     2941 gggatcgagg aaaacctgta cttccaatcc ggttctggaa tggtttccaa aggagaggag
     3001 gataatatgg ctataattaa agagtttatg cggttcaagg tgcatatgga gggatctgtg
     3061 aacggtcacg agttcgaaat cgagggcgaa ggcgaggggc gaccctatga agggacacag
     3121 acggccaaac tgaaggtgac caagggtggc cccctgcctt tcgcctggga catcctgtca
     3181 ccccagttta tgtacggctc taaggcttac gttaagcacc ctgccgatat tccggactac
     3241 ttgaaactga gcttccctga agggttcaag tgggaacggg taatgaattt tgaggatggc
     3301 ggtgtagtca cagttaccca ggacagctct cttcaggacg gagaatttat ctataaggtt
     3361 aaacttcggg gcactaactt cccatcagac ggccccgtga tgcagaagaa aactatgggc
     3421 tgggaagcca gctcagaacg catgtatccc gaggacggag cactgaaggg agaaatcaag
     3481 cagcgactca agctgaagga tgggggacat tatgatgctg aggtcaaaac cacctataag
     3541 gccaagaaac ccgttcagct tcctggggca tacaacgtga atatcaaact ggatattaca
     3601 tcccataacg aagactatac catcgtggaa cagtacgagc gggccgaggg cagacatagc
     3661 acagggggca tggatgaatt gtacaagtac acggattact caccgacatc accagcctat
     3721 gagccacgct cccctggggg ctatacaccc cagagcccct cctactcccc tacttcacct
     3781 tcctactccc caacctctcc atcttactct ccaaccagtc ccaactacag ccctacctct
     3841 cctagctact cgcccacctc ccctagctac tcgccaacct ctccttccta ctcccccacc
     3901 tctccaagct attccccaac ctctcctagc tactccccaa cctctccaag ctattctcca
     3961 acatcaccta gctattctcc aacttctccc agctactcac cgacatctcc tagctactcc
     4021 ccaacttctc ccagctactc accaacttca ccaagctatt ctcccacctc ccccagctac
     4081 tcaccgacat ctcccagcta ctcaccaact tctccaagct actcaccaac ttctccaagc
     4141 tactcaccca ccagccctaa ctattctcca actagtccca actatacccc gacatcaccc
     4201 agctacagcc caacctcacc cagttactca cctacaagtc ccaactatac acccaccagc
     4261 cctaattaca gcccaacctc tccaagctat tccccaacct cacccagtta ttcccccacc
     4321 tcaccaagct actccccctc cagcccacga tatacaccac agtctccaac ctacacacca
     4381 agctcaccaa gctacagtcc cagctcacca agctacagcc ccacttcacc caagtacacc
     4441 ccaactagtc cttcctacag tcccagctca ccagagtaca ccccagcttc tcccaaatac
     4501 tcacctacaa gccctaaata ttcacccact tctcccaagt attctcctac cagccccact
     4561 tactcaccta ccaccccaaa atattctcca acctccccga catactcacc aacctctcca
     4621 gtctataccc cgacctctcc caagtactcc ccaaccagcc ctacctactc gcccacttct
     4681 cccaagtact cgcccaccag tcccacctac tcacccacct ctcccaaggg ctccacctac
     4741 tctcccactt ctcctggcta ctcacccact agccccacct acagcctcac cagcccagcc
     4801 atcagcccag atgacagcga tgaggagaac gttgcggccg cactcgagtc tggtaaagaa
     4861 accgctgctg cgaaatttga acgccagcac atggactcgt ctactagcgc agcttaatta
     4921 acctaggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
     4981 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
     5041 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     5101 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     5161 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     5221 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
     5281 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     5341 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     5401 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5461 attttaacaa aatattaacg tttacaattt ctggcggcac gatggcatga gattatcaaa
     5521 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
     5581 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
     5641 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
     5701 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
     5761 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
     5821 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
     5881 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
     5941 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
     6001 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
     6061 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
     6121 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
     6181 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
     6241 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
     6301 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
     6361 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
     6421 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     6481 atcatgattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6541 tttagaaaaa taaacaaata ggtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6601 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6661 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6721 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6781 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6841 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6901 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6961 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     7021 cagcgtgagc tatgagaaag cgccacgctt cccgaag
//
