LOCUS       sequence-364587-        5941 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial Expression of 6xHis Sirius-GFP Fusion Protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5941)
  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 5941)
  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-mEGFP
FEATURES             Location/Qualifiers
     source          1..5941
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     93..112
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     346..363
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    378..517
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(443..462)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     603..625
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(619..810)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(1382..2464)
                     /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     2426..2445
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(2465..2542)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     2748..2767
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     2855..2874
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2855..2873
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2874..2898
                     /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             2913..2935
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2945..2962
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2972..2992
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(3002..3004,3005..3712)
                     /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(3044..3065)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     3652..3673
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3761..3805
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3843..3861)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3857..3904
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3941..4396
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4028..4047)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4238..4259
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(4511..5302,5303..5371))
                     /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     5134..5153
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5372..5464)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      join(5545..5941,1..192)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
       61 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
      121 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
      181 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
      241 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
      301 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
      361 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
      421 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
      481 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
      541 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
      601 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
      661 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
      721 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
      781 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
      841 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
      901 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
      961 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     1021 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     1081 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     1141 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     1201 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     1261 gatcatgcta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     1321 gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg
     1381 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1441 acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg
     1501 agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt
     1561 ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat
     1621 aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca
     1681 gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca
     1741 tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg
     1801 cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat
     1861 gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga
     1921 tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg
     1981 agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat
     2041 tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca
     2101 gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc
     2161 ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg
     2221 ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca
     2281 acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca
     2341 tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc
     2401 gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt
     2461 tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg
     2521 ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt
     2581 aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca
     2641 tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg
     2701 aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg
     2761 atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg
     2821 tagaggatcg agatcgatct cgatcccgcg aaattaatac gactcactat aggggaattg
     2881 tgagcggata acaattcccc tctagaaata attttgttta actttaagaa ggagatatac
     2941 catgcaccat caccatcacc atgggatcga ggaaaacctg tacttccaat ccggttctgg
     3001 agtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg
     3061 cgacgtaaac ggccacaagt tcagcgtgcg cggcgagggc gagggcgatg ccaccaacgg
     3121 caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct
     3181 cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca
     3241 gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatctcctt
     3301 caaggacgac ggcacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt
     3361 gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa
     3421 gctggagtac aacttcaaca gccacaacgt ctatatcacg gccgacaagc agaagaacgg
     3481 catcaaggcg aacttcaaga tccgccacaa cgtcgaggac ggcagcgtgc agctcgccga
     3541 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     3601 cctgagcacc cagtccaagc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     3661 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acacggcgcg
     3721 cctgcaggtc gacaagcttg cggccgcact cgagtctggt aaagaaaccg ctgctgcgaa
     3781 atttgaacgc cagcacatgg actcgtctac tagcgcagct taattaacct aggctgctgc
     3841 caccgctgag caataactag cataacccct tggggcctct aaacgggtct tgaggggttt
     3901 tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg tagcggcgca
     3961 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4021 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4081 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4141 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     4201 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     4261 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     4321 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     4381 ttaacgttta caatttctgg cggcacgatg gcatgagatt atcaaaaagg atcttcacct
     4441 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     4501 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     4561 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     4621 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     4681 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     4741 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     4801 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     4861 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     4921 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     4981 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     5041 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     5101 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     5161 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     5221 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     5281 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     5341 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatca tgattgaagc
     5401 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5461 caaataggtc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     5521 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     5581 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     5641 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     5701 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     5761 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     5821 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     5881 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     5941 a
//
