LOCUS       sequence-349907-        6666 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 6666)
  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 6666)
  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-c007
FEATURES             Location/Qualifiers
     source          1..6666
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             4..21
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             25..537
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             544..564
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             1443..1460
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1513..1531)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1527..1574
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1611..2066
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1698..1717)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1908..1929
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2158..2973)
                     /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(2158..2973)
                     /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     2881..2900
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2965..2984
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3095..3683
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3584..3603
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3837..3854
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3869..4008
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3934..3953)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4094..4116
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4110..4301)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLQDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5110..6192)
                     /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     6154..6173
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6193..6270)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6476..6495
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6579..6598
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6579..6597
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6598..6622
                     /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             6637..6659
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 atgcaccatc atcatcatca ttctatggtc ttcacactcg aagatttcgt tggggactgg
       61 cgacagacag ccggctacaa cctggaccaa gtccttgaac agggaggtgt gtccagtttg
      121 tttcagaatc tcggggtgtc cgtaactccg atccaaagga ttgtcctgag cggtgaaaat
      181 gggctgaaga tcgacatcca tgtcatcatc ccgtatgaag gtctgagcgg cgaccaaatg
      241 ggccagatcg aaaaaatttt taaggtggtg taccctgtgg acgatcatca ctttaaggtg
      301 atcctgcact atggcacact ggtaatcgac ggggttacgc cgaacatgat cgactatttc
      361 ggacggccgt atgaaggcat cgccgtgttc gacggcaaaa agatcactgt aacagggacc
      421 ctgtggaacg gcaacaaaat tatcgacgag cgcctgatca accccgacgg ctccctgctg
      481 ttccgagtaa ccatcaacgg agtgaccggc tggcggctgt gcgaacgcat tctggcgggt
      541 accgagaacc tgtacttcca atccatgctg cgtaacaaag gtagcgtgct gatcccgggt
      601 ctggttgaag gcagcaccaa gcgtaagcgt gttctgagcc cggaggaact ggaagaccgt
      661 cagaagaaac gtcaagaggc ggcggcgaaa cgtcgtcgtc tgattgagga aaagaaacgt
      721 cagaaggaag aggcggaaca caagaaaaag atggcgtggt gggagagcaa caactaccaa
      781 agcttctgcc tgccgagcga ggaaagcgaa ccggaggacc tggaaaacgg cgaggaaagc
      841 agcgcggagc tggattacca ggacccggat gcgaccagcc tgaaatatgt tagcggtgat
      901 gttacccacc cgcaagcggg tgcggaggat gcgctgatcg tgcactgcgt tgacgatagc
      961 ggtcactggg gtcgtggtgg cctgttcacc gcgctggaaa aacgtagcgc ggagccgcgt
     1021 aagatttatg aactggcggg caaaatgaag gatctgagcc tgggtggcgt gctgctgttt
     1081 ccggttgacg ataaagagag ccgtaacaag ggtcaggacc tgctggcgct gatcgtggcg
     1141 caacaccgtg atcgtagcaa cgttctgagc ggcatcaaga tggcggcgct ggaggaaggt
     1201 ctgaaaaaga ttttcctggc ggcgaaaaag aaaaaggcga gcgtgcacct gccgcgtatc
     1261 ggtcacgcga ccaaaggctt taactggtac ggtaccgagc gtctgattcg taagcacctg
     1321 gcggcgcgtg gtatcccgac ctatatttac tatttcccgc gtagcaaata ggacggtctc
     1381 cagtaaaggt ggatacggat ccgaattcga gctccgtcga caagcttgcg gccgcactcg
     1441 agcaccacca ccaccaccac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt
     1501 tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct
     1561 tgaggggttt tttgctgaaa ggaggaacta tatccggatt ggcgaatggg acgcgccctg
     1621 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     1681 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     1741 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     1801 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     1861 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     1921 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     1981 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     2041 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     2101 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgaa ttaattctta
     2161 gaaaaactca tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc
     2221 atatttttga aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag
     2281 gatggcaaga tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat
     2341 taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga
     2401 atccggtgag aatggcaaaa gtttatgcat ttctttccag acttgttcaa caggccagcc
     2461 attacgctcg tcatcaaaat cactcgcatc aaccaaaccg ttattcattc gtgattgcgc
     2521 ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa ttacaaacag gaatcgaatg
     2581 caaccggcgc aggaacactg ccagcgcatc aacaatattt tcacctgaat caggatattc
     2641 ttctaatacc tggaatgctg ttttcccggg gatcgcagtg gtgagtaacc atgcatcatc
     2701 aggagtacgg ataaaatgct tgatggtcgg aagaggcata aattccgtca gccagtttag
     2761 tctgaccatc tcatctgtaa catcattggc aacgctacct ttgccatgtt tcagaaacaa
     2821 ctctggcgca tcgggcttcc catacaatcg atagattgtc gcacctgatt gcccgacatt
     2881 atcgcgagcc catttatacc catataaatc agcatccatg ttggaattta atcgcggcct
     2941 agagcaagac gtttcccgtt gaatatggct cataacaccc cttgtattac tgtttatgta
     3001 agcagacagt tttattgttc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3061 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     3121 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     3181 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     3241 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     3301 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     3361 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     3421 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     3481 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     3541 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     3601 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     3661 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     3721 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     3781 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     3841 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     3901 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     3961 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4021 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4081 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     4141 gcgaggcagc tgcggtaaag ctcatcagcg tggtcctgaa gcgattcaca gatgtctgcc
     4201 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     4261 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     4321 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     4381 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     4441 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     4501 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     4561 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     4621 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     4681 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     4741 gacaggagca cgatcatgcg cacccgtggg gccgccatgc cggcgataat ggcctgcttc
     4801 tcgccgaaac gtttggtggc gggaccagtg acgaaggctt gagcgagggc gtgcaagatt
     4861 ccgaataccg caagcgacag gccgatcatc gtcgcgctcc agcgaaagcg gtcctcgccg
     4921 aaaatgaccc agagcgctgc cggcacctgt cctacgagtt gcatgataaa gaagacagtc
     4981 ataagtgcgg cgacgatagt catgccccgc gcccaccgga aggagctgac tgggttgaag
     5041 gctctcaagg gcatcggtcg agatcccggt gcctaatgag tgagctaact tacattaatt
     5101 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     5161 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     5221 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     5281 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     5341 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     5401 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     5461 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     5521 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     5581 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     5641 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     5701 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     5761 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     5821 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     5881 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     5941 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     6001 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     6061 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     6121 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     6181 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     6241 gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact
     6301 cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga
     6361 atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac
     6421 ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga
     6481 tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc
     6541 gtccggcgta gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga
     6601 attgtgagcg gataacaatt cccctctaga aataattttg tttaacttta agaaggagat
     6661 atacat
//
