LOCUS       sequence-349927-        6714 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of recombinant fusion protein in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6714)
  AUTHORS   Sowa ST, Galera-Prat A, Wazir S, Alanen HI, Maksimainen MM, Lehtio L
  TITLE     A molecular toolbox for ADP-ribosyl binding proteins.
  JOURNAL   Cell Rep Methods. 2021 Dec 20;1(8):100121. doi: 
            10.1016/j.crmeth.2021.100121. Epub 2021 Nov 11.
  PUBMED    34786571
REFERENCE   2  (bases 1 to 6714)
  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: "Cell Rep 
            Methods."; date: "2021-12-20"; volume: "1(8):100121. doi"; pages: " 
            10.1016/j.crmeth.2021.100121. Epub 2021 Nov 11"
COMMENT     Sequence Label: MacroH2A1.2-c002
FEATURES             Location/Qualifiers
     source          1..6714
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4..21
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             join(22..24,25..735)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIKA
                     HFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(64..85)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(325..344)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     672..693
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             760..780
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1491..1508
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1561..1579)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1575..1622
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1659..2114
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1746..1765)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1956..1977
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2206..3021)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(2206..3021)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2929..2948
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3013..3032
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3143..3731
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3632..3651
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3885..3902
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3917..4056
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3982..4001)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4142..4164
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4158..4349)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5158..6240)
                     /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     6202..6221
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6241..6318)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6524..6543
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6627..6646
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6627..6645
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6646..6670
                     /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             6685..6707
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgcaccatc atcatcatca tgtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc
       61 atcctggtcg agctggacgg cgacgtaaac ggccacaggt tcagcgtgtc cggcgagggc
      121 gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg
      181 cccgtgccct ggcccaccct cgtgaccacc ctgacctggg gcgtgcagtg cttcgcccgc
      241 taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc
      301 caggagcgta ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag
      361 ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac
      421 ggcaacatcc tggggcacaa gctggagtac aacgccatca gcgacaacgt ctatatcacc
      481 gccgacaagc agaagaacgg catcaaggcc cacttcaaga tccgccacaa catcgaggac
      541 ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
      601 ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag
      661 aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg
      721 gacgagctgt acaagtcttc tggtgtagat ctgggtaccg agaacctgta cttccaatcc
      781 atgcaaggcg aggtttctaa ggcggcgagc gcggacagca ccaccgaagg taccccggcg
      841 gatggcttca ccgtgctgag caccaagagc ctgtttctgg gtcaaaaact gaacctgatc
      901 cacagcgaga ttagcaacct ggcgggcttc gaggttgaag cgatcattaa cccgaccaac
      961 gcggacatcg atctgaagga cgatctgggt aacaccctgg aaaagaaagg tggcaaagag
     1021 tttgtggaag cggttctgga gctgcgtaag aaaaacggtc cgctggaagt ggcgggtgcg
     1081 gcggttagcg cgggtcatgg tctgccggcg aagttcgtga ttcactgcaa cagcccggtt
     1141 tggggtgcgg acaaatgcga ggaactgctg gaaaagaccg tgaaaaactg cctggcgctg
     1201 gcggacgata agaaactgaa aagcatcgcg ttcccgagca ttggtagcgg ccgtaacggc
     1261 tttccgaagc agaccgcggc gcaactgatc ctgaaagcga ttagcagcta cttcgttagc
     1321 accatgagca gcagcatcaa gaccgtgtac ttcgttctgt ttgacagcga gagcatcggt
     1381 atttatgtgc aggaaatggc gaaactggat gcgaactaag acggtctcca gtaaaggtgg
     1441 atacggatcc gaattcgagc tccgtcgaca agcttgcggc cgcactcgag caccaccacc
     1501 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
     1561 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
     1621 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
     1681 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     1741 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     1801 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     1861 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1921 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     1981 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2041 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     2101 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     2161 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     2221 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     2281 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     2341 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     2401 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     2461 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     2521 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     2581 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     2641 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     2701 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     2761 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     2821 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     2881 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     2941 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     3001 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     3061 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     3121 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     3181 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     3241 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     3301 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     3361 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     3421 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     3481 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     3541 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     3601 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     3661 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     3721 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     3781 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     3841 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3901 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3961 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     4021 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     4081 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4141 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     4201 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     4261 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     4321 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     4381 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     4441 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     4501 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     4561 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     4621 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     4681 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     4741 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     4801 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     4861 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     4921 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     4981 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     5041 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     5101 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     5161 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     5221 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     5281 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     5341 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     5401 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     5461 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     5521 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     5581 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     5641 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     5701 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     5761 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     5821 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     5881 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     5941 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     6001 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     6061 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     6121 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     6181 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     6241 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     6301 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     6361 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     6421 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     6481 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     6541 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga
     6601 ggatcgagat ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga
     6661 taacaattcc cctctagaaa taattttgtt taactttaag aaggagatat acat
//
