LOCUS       sequence-349882-        6348 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 6348)
  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 6348)
  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: APLF-c001
FEATURES             Location/Qualifiers
     source          1..6348
                     /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             1125..1142
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1195..1213)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1209..1256
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1293..1748
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1380..1399)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1590..1611
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1840..2655)
                     /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(1840..2655)
                     /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     2563..2582
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2647..2666
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2777..3365
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3266..3285
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3519..3536
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3551..3690
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3616..3635)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3776..3798
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3792..3983)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4792..5874)
                     /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     5836..5855
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5875..5952)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6158..6177
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6261..6280
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6261..6279
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6280..6304
                     /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             6319..6341
                     /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 atgaaagcca cagattccgt tttacaaggt tcagagggta ataaagtgaa acgtacctca
      841 tgcatgtatg gggcgaattg ttaccgcaaa aaccccgtcc acttccaaca cttcagccat
      901 ccaggggaca gtgactacgg aggagtgcaa attgtaggcc aggacgaaac agatgaccgc
      961 cctgagtgtc cgtatggtcc gtcctgctac cgtaaaaatc cacaacataa gatcgaatat
     1021 cgccataata cacttcctgt acgcaacgtc taggacggtc tccagtaaag gtggatacgg
     1081 atccgaattc gagctccgtc gacaagcttg cggccgcact cgagcaccac caccaccacc
     1141 actgagatcc ggctgctaac aaagcccgaa aggaagctga gttggctgct gccaccgctg
     1201 agcaataact agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga
     1261 aaggaggaac tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
     1321 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     1381 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     1441 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     1501 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     1561 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     1621 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     1681 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
     1741 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1801 taaatacatt caaatatgta tccgctcatg aattaattct tagaaaaact catcgagcat
     1861 caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg
     1921 tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta
     1981 tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa
     2041 aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa
     2101 aagtttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa
     2161 atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac
     2221 gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac
     2281 tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc
     2341 tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg
     2401 cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt
     2461 aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt
     2521 cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata
     2581 cccatataaa tcagcatcca tgttggaatt taatcgcggc ctagagcaag acgtttcccg
     2641 ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt
     2701 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     2761 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     2821 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     2881 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     2941 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3001 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3061 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3121 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3181 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3241 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3301 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3361 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3421 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     3481 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     3541 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3601 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     3661 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     3721 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     3781 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     3841 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     3901 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     3961 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     4021 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     4081 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     4141 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     4201 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     4261 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     4321 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     4381 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     4441 cgcacccgtg gggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     4501 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     4561 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     4621 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     4681 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     4741 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     4801 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4861 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     4921 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     4981 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     5041 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     5101 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     5161 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     5221 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     5281 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     5341 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     5401 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     5461 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     5521 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     5581 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     5641 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     5701 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     5761 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     5821 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     5881 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     5941 ttcgatggtg tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc
     6001 ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga
     6061 tggcgcccaa cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc
     6121 tcatgagccc gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc
     6181 cagcaaccgc acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg
     6241 agatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa
     6301 ttcccctcta gaaataattt tgtttaactt taagaaggag atatacat
//
