LOCUS       sequence-349906-        6699 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 6699)
  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 6699)
  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: MacroH2A.2-c002
FEATURES             Location/Qualifiers
     source          1..6699
                     /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             1476..1493
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1546..1564)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1560..1607
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1644..2099
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1731..1750)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1941..1962
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2191..3006)
                     /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(2191..3006)
                     /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     2914..2933
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2998..3017
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3128..3716
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3617..3636
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3870..3887
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3902..4041
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3967..3986)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4127..4149
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4143..4334)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5143..6225)
                     /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     6187..6206
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6226..6303)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6509..6528
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6612..6631
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6612..6630
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6631..6655
                     /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             6670..6692
                     /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 agaacttgta cttccaatcc
      781 atgaaagaag gaacttcaaa ttccacctct gaagatgggc caggggatgg attcaccatt
      841 ctgtcttcta agagccttgt tctgggacag aagctgtcct taacccagag tgacatcagc
      901 catattggct ccatgagagt ggagggcatt gtccacccaa ccacagccga aattgacctc
      961 aaagaagata taggtaaagc cttggaaaag gctgggggaa aagagttctt ggaaacggta
     1021 aaggagcttc gcaaatccca aggccctttg gaagtcgccg aagccgccgt cagccaatcc
     1081 agtggactcg cagccaaatt tgtcatccac tgtcacatcc ctcagtgggg ctccgacaaa
     1141 tgtgaagaac agcttgaaga gaccatcaaa aactgcctgt cagcggcgga ggacaagaag
     1201 ctaaagtccg tcgcgttccc gcctttcccc agcggcagaa actgctttcc caaacagact
     1261 gcggcccagg tgaccctcaa agccatctca gcccactttg atgactcgag cgcgtcctcg
     1321 ctgaagaacg tgtacttcct gctcttcgac agcgagagca tcggcatcta cgtgcaggag
     1381 atggccaagc tcgacgccaa gtaagacggt ctccagtaaa ggtggatacg gatccgaatt
     1441 cgagctccgt cgacaagctt gcggccgcac tcgagcacca ccaccaccac cactgagatc
     1501 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     1561 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1621 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1681 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1741 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1801 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1861 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1921 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1981 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2041 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     2101 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     2161 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     2221 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     2281 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     2341 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     2401 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     2461 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     2521 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     2581 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     2641 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     2701 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     2761 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     2821 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     2881 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     2941 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     3001 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     3061 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3121 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3181 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3241 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     3301 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     3361 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     3421 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     3481 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3541 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3601 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3661 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3721 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3781 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3841 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3901 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     3961 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     4021 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     4081 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     4141 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     4201 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     4261 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     4321 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     4381 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     4441 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     4501 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     4561 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     4621 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     4681 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     4741 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     4801 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     4861 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     4921 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     4981 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     5041 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     5101 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     5161 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5221 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     5281 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     5341 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     5401 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     5461 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     5521 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     5581 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     5641 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     5701 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     5761 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     5821 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     5881 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     5941 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     6001 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     6061 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     6121 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     6181 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     6241 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     6301 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     6361 ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca
     6421 acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc
     6481 cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg
     6541 cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga
     6601 tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca attcccctct
     6661 agaaataatt ttgtttaact ttaagaagga gatatacat
//
