LOCUS       sequence-349915-        6882 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 6882)
  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 6882)
  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: ALC1-c005
FEATURES             Location/Qualifiers
     source          1..6882
                     /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             1659..1676
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1729..1747)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1743..1790
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1827..2282
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1914..1933)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2124..2145
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2374..3189)
                     /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(2374..3189)
                     /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     3097..3116
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3181..3200
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3311..3899
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3800..3819
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4053..4070
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4085..4224
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4150..4169)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4310..4332
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4326..4517)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5326..6408)
                     /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     6370..6389
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6409..6486)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6692..6711
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6795..6814
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6795..6813
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6814..6838
                     /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             6853..6875
                     /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 atgctgcgta acaaaggtag cgtgctgatc ccgggtctgg ttgaaggcag caccaagcgt
      841 aagcgtgttc tgagcccgga ggaactggaa gaccgtcaga agaaacgtca agaggcggcg
      901 gcgaaacgtc gtcgtctgat tgaggaaaag aaacgtcaga aggaagaggc ggaacacaag
      961 aaaaagatgg cgtggtggga gagcaacaac taccaaagct tctgcctgcc gagcgaggaa
     1021 agcgaaccgg aggacctgga aaacggcgag gaaagcagcg cggagctgga ttaccaggac
     1081 ccggatgcga ccagcctgaa atatgttagc ggtgatgtta cccacccgca agcgggtgcg
     1141 gaggatgcgc tgatcgtgca ctgcgttgac gatagcggtc actggggtcg tggtggcctg
     1201 ttcaccgcgc tggaaaaacg tagcgcggag ccgcgtaaga tttatgaact ggcgggcaaa
     1261 atgaaggatc tgagcctggg tggcgtgctg ctgtttccgg ttgacgataa agagagccgt
     1321 aacaagggtc aggacctgct ggcgctgatc gtggcgcaac accgtgatcg tagcaacgtt
     1381 ctgagcggca tcaagatggc ggcgctggag gaaggtctga aaaagatttt cctggcggcg
     1441 aaaaagaaaa aggcgagcgt gcacctgccg cgtatcggtc acgcgaccaa aggctttaac
     1501 tggtacggta ccgagcgtct gattcgtaag cacctggcgg cgcgtggtat cccgacctat
     1561 atttactatt tcccgcgtag caaataggac ggtctccagt aaaggtggat acggatccga
     1621 attcgagctc cgtcgacaag cttgcggccg cactcgagca ccaccaccac caccactgag
     1681 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     1741 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     1801 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     1861 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     1921 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     1981 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2041 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2101 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2161 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2221 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     2281 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     2341 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     2401 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     2461 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     2521 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     2581 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     2641 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     2701 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     2761 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     2821 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     2881 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     2941 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     3001 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     3061 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     3121 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     3181 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     3241 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3301 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     3361 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     3421 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     3481 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     3541 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3601 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3661 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3721 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3781 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3841 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3901 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3961 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4021 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4081 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     4141 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     4201 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4261 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4321 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     4381 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     4441 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     4501 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     4561 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     4621 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     4681 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     4741 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     4801 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     4861 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     4921 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     4981 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     5041 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     5101 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     5161 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     5221 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     5281 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     5341 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5401 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     5461 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     5521 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     5581 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     5641 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     5701 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     5761 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     5821 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     5881 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     5941 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     6001 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     6061 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     6121 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     6181 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     6241 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     6301 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     6361 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     6421 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc gccattcgat
     6481 ggtgtccggg atctcgacgc tctcccttat gcgactcctg cattaggaag cagcccagta
     6541 gtaggttgag gccgttgagc accgccgccg caaggaatgg tgcatgcaag gagatggcgc
     6601 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     6661 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     6721 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg atcgagatct
     6781 cgatcccgcg aaattaatac gactcactat aggggaattg tgagcggata acaattcccc
     6841 tctagaaata attttgttta actttaagaa ggagatatac at
//
