LOCUS       sequence-364591-        6508 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of 6xHis NFYC IDR Sirius-GFP Fusion Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6508)
  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 6508)
  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-NFYC-IDR-mEGFP
FEATURES             Location/Qualifiers
     source          1..6508
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     301..320
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     554..571
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    586..725
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(651..670)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     811..833
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(827..1018)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1590..2672)
                     /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     2634..2653
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2673..2750)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2956..2975
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3063..3082
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3063..3081
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3082..3106
                     /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             3121..3143
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3153..3170
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3180..3200
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(3210..3212,3213..3920)
                     /codon_start=1
                     /product="enhanced GFP (EGFP) derivative that exhibits 
                     robust folding and enhanced photostability (Zhong et al., 
                     2019)"
                     /label=SiriusGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     primer_bind     complement(3252..3273)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3860..3881
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4536..4580
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(4618..4636)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4632..4679
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4716..5171
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4803..4822)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5013..5034
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(5286..6077,6078..6146))
                     /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     5909..5928
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6147..6239)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(6320..6508,1..400)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
       61 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
      121 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
      181 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
      241 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
      301 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
      361 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
      421 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
      481 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
      541 aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt
      601 tctccttacg catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc
      661 tctgatgccg catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg
      721 ctgcgccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
      781 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
      841 cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg
      901 attcacagat gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta
      961 atgtctggct tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg
     1021 atgcctccgt gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga
     1081 ggatgctcac gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg
     1141 gtaaacaact ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc
     1201 agcgcttcgt taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc
     1261 agatccggaa cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac
     1321 ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc
     1381 ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc
     1441 ctagccgggt cctcaacgac aggagcacga tcatgctagt catgccccgc gcccaccgga
     1501 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     1561 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     1621 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     1681 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     1741 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     1801 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     1861 actaccgaga tgtccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     1921 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     1981 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     2041 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     2101 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     2161 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     2221 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     2281 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     2341 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     2401 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     2461 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     2521 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     2581 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     2641 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     2701 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     2761 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     2821 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
     2881 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
     2941 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
     3001 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atcgatctcg atcccgcgaa
     3061 attaatacga ctcactatag gggaattgtg agcggataac aattcccctc tagaaataat
     3121 tttgtttaac tttaagaagg agatatacca tgcaccatca ccatcaccat gggatcgagg
     3181 aaaacctgta cttccaatcc ggttctggag tgagcaaggg cgaggagctg ttcaccgggg
     3241 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgcgcg
     3301 gcgagggcga gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg
     3361 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     3421 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     3481 gctacgtcca ggagcgcacc atctccttca aggacgacgg cacctacaag acccgcgccg
     3541 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     3601 aggaggacgg caacatcctg gggcacaagc tggagtacaa cttcaacagc cacaacgtct
     3661 atatcacggc cgacaagcag aagaacggca tcaaggcgaa cttcaagatc cgccacaacg
     3721 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     3781 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg agcaaagacc
     3841 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     3901 tcggcatgga cgagctgtac acggaccctg tccagtacta cttcacgctg gctcagcagc
     3961 ccactgctgt ccaggtccag ggacagcagc aaggccagca gaccaccagt tctacgacca
     4021 ccatccagcc tggacagatc atcattgcac agcctcagca gggtcagacc acacccgtga
     4081 ccatgcaggt tggagaaggt cagcaggtgc agattgtaca ggcccaacct cagggtcagg
     4141 cccagcagac ccagagtggc actggacaga ccatgcaggt gatgcagcag atcattacca
     4201 acacaggaga gatccagcag atcccggtgc agctgaatgc tggccagctg caatatatcc
     4261 gattagccca gcctgtatca ggcacccaag ttgtccaagg acagatccag acacttgcta
     4321 ccaatgccca acagatcaca cagacagagg tccagcaagg acagcagcag ttcagccagt
     4381 tcacagatgg acagcagctg taccagatcc agcaagtcac catgcctgca ggccaagacc
     4441 tcgcccagcc catgtttatt cagtcagcca atcagccctc tgatgggcag accccccagg
     4501 tgactggaga cattgcggcc gcactcgagt ctggtaaaga aaccgctgct gcgaaatttg
     4561 aacgccagca catggactcg tctactagcg cagcttaatt aacctaggct gctgccaccg
     4621 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     4681 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     4741 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     4801 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     4861 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     4921 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     4981 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5041 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     5101 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5161 gtttacaatt tctggcggca cgatggcatg agattatcaa aaaggatctt cacctagatc
     5221 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5281 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5341 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5401 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5461 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5521 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5581 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5641 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5701 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5761 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5821 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5881 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5941 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6001 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6061 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6121 gcgacacgga aatgttgaat actcatactc ttcctttttc aatcatgatt gaagcattta
     6181 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6241 aggtcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6301 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6361 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6421 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     6481 cgtagttagg ccaccacttc aagaactc
//
