LOCUS       sequence-349923-        6447 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 6447)
  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 6447)
  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: AF1521-c003
FEATURES             Location/Qualifiers
     source          1..6447
                     /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             1224..1241
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1294..1312)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1308..1355
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1392..1847
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1479..1498)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1689..1710
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1939..2754)
                     /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(1939..2754)
                     /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     2662..2681
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2746..2765
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2876..3464
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3365..3384
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3618..3635
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3650..3789
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3715..3734)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3875..3897
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3891..4082)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLQDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4891..5973)
                     /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     5935..5954
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5974..6051)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6257..6276
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6360..6379
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6360..6378
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6379..6403
                     /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             6418..6440
                     /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 atccatgaag cacatggagg tactgtttga ggctaaggtg
      601 ggggatatta cgttgcgtct tgcacagggg gacatcacac agtaccccgc taaagccatt
      661 gtgaatgcgg ccaatgagcg tttggaacat ggcggaggtg tcgcttacgc tatcgctaag
      721 gcgtgcgctg gcgatgcggg tttatatacg gaaatttcaa aaaaggctat gcgtgagcaa
      781 tttggtcgcg actgtattga ccatggtgag gtagttgtta caccggcaat gaacttagag
      841 gaacgcggta tcaagtacgt gcttcacacc gtaggccccg tatgcggagg catgtggtcc
      901 ggagagctta aagaaaaact ttacaaagcc ttccttggtc cgttggaaaa agctgaagag
      961 atgggagtag aaagcatcgc ctttcctgca gtctccgcag gtattcgcgg ctgcgatttg
     1021 gaaaaggtcg ttgaaacatt ccttgaagcg gtcaaagact tcaaggggag cgcagtgaag
     1081 gaggttgcgt tggtcatcta tgaccgtaaa tccgctgagg ttgcattaaa agtttttgaa
     1141 cgcagtctgt aggacggtct ccagtaaagg tggatacgga tccgaattcg agctccgtcg
     1201 acaagcttgc ggccgcactc gagcaccacc accaccacca ctgagatccg gctgctaaca
     1261 aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta gcataacccc
     1321 ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact atatccggat
     1381 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     1441 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     1501 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     1561 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     1621 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     1681 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     1741 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     1801 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     1861 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     1921 ccgctcatga attaattctt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     1981 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     2041 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     2101 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     2161 atcaccatga gtgacgactg aatccggtga gaatggcaaa agtttatgca tttctttcca
     2221 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     2281 gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt taaaaggaca
     2341 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     2401 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     2461 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     2521 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     2581 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     2641 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     2701 gttggaattt aatcgcggcc tagagcaaga cgtttcccgt tgaatatggc tcataacacc
     2761 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgaccaaa atcccttaac
     2821 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     2881 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     2941 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     3001 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     3061 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     3121 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     3181 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     3241 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     3301 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     3361 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     3421 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     3481 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     3541 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     3601 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     3661 attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc tcagtacaat
     3721 ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg tgactgggtc
     3781 atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3841 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3901 tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc gtggtcctga
     3961 agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt ctccagaagc
     4021 gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc ctgtttggtc
     4081 actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc gatgaaacga
     4141 gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact ggaacgttgt
     4201 gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac tcagggtcaa
     4261 tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca gcatcctgcg
     4321 atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag actttacgaa
     4381 acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt gcagcagcag
     4441 tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag gcaaccccgc
     4501 cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg ggccgccatg
     4561 ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt gacgaaggct
     4621 tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat cgtcgcgctc
     4681 cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg tcctacgagt
     4741 tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg cgcccaccgg
     4801 aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg tgcctaatga
     4861 gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     4921 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     4981 cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc ccttcaccgc
     5041 ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca ggcgaaaatc
     5101 ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat cgtcgtatcc
     5161 cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc gcattgcgcc
     5221 cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct cattcagcat
     5281 ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt ccgctatcgg
     5341 ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac gcgccgagac
     5401 agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga ccagatgctc
     5461 cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg gtgtctggtc
     5521 agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag caatggcatc
     5581 ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga gaagattgtg
     5641 caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca ccacgctggc
     5701 acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg cgtgcagggc
     5761 cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt gttgtgccac
     5821 gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc gcgttttcgc
     5881 agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga caccggcata
     5941 ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt gactctcttc
     6001 cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt ccgggatctc
     6061 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     6121 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     6181 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     6241 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     6301 cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt
     6361 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     6421 gtttaacttt aagaaggaga tatacat
//
