LOCUS       sequence-349930-        7131 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 7131)
  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 7131)
  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: ARH3-c002
FEATURES             Location/Qualifiers
     source          1..7131
                     /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             1908..1925
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1978..1996)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1992..2039
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2076..2531
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2163..2182)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2373..2394
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2623..3438)
                     /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(2623..3438)
                     /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     3346..3365
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3430..3449
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3560..4148
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4049..4068
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4302..4319
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4334..4473
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4399..4418)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4559..4581
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4575..4766)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5575..6657)
                     /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     6619..6638
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6658..6735)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6941..6960
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7044..7063
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7044..7062
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7063..7087
                     /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             7102..7124
                     /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 atggctggag cggcacgtag cctgagccgc ttccgtggct gcttggctgg tgctctgtta
      841 ggagattgcg tagggtcctt ttacgaagct catgacaccg tcgatttaac gagcgtactt
      901 cgtcacgttc aaagcctgga acctgatccc gggacgcctg gttctgagcg tactgaagca
      961 ttatattaca ccgacgatac tgccatggcc cgcgcccttg ttcagtctct tttagctaag
     1021 gaggcgttcg acgaggtgga catggctcac cgtttcgcgc aggaatacaa gaaagacccc
     1081 gaccgtggat acggtgcagg tgtagtgacc gtatttaaga aacttttaaa cccgaaatgt
     1141 cgtgacgttt ttgaaccagc ccgtgctcaa ttcaatggta aaggttcgta tggcaacggt
     1201 ggggctatgc gtgtcgcagg catctcactg gcgtactctt cggttcaaga tgtccagaag
     1261 tttgcccgtt tatcagcgca attgacacac gcttcaagcc ttgggtacaa cggagccatc
     1321 ttacaggcac ttgccgtaca tttagcattg cagggggagt caagctcaga gcatttcttg
     1381 aaacaactgc ttggccacat ggaggatttg gaaggagatg cacaatccgt acttgacgcc
     1441 cgtgaactgg gcatggaaga gcgtccctac tcttcccgcc ttaagaagat tggagagttg
     1501 ctggatcagg cttcggtcac tcgcgaggaa gttgtatcag aattgggaaa cgggatcgct
     1561 gcgtttgagt ctgtaccaac tgccatctac tgctttctgc gctgcatgga accggaccca
     1621 gagatcccct ctgcgttcaa ctccctgcaa cgcacgctga tctatagcat ttcactggga
     1681 ggcgataccg atactattgc taccatggct ggtgccattg ctggcgccta ttatggaatg
     1741 gatcaagtgc cggaaagctg gcagcaaagc tgcgaggggt atgaggagac tgacattctt
     1801 gcgcagtcct tacaccgtgt ttttcaaaag tcctaagacg gtctccagta aaggtggata
     1861 cggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc
     1921 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     1981 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     2041 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     2101 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     2161 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     2221 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2281 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     2341 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2401 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2461 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2521 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2581 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     2641 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     2701 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     2761 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     2821 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     2881 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     2941 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     3001 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     3061 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     3121 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     3181 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     3241 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     3301 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     3361 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     3421 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     3481 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3541 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3601 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3661 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3721 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3781 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3841 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3901 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3961 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     4021 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     4081 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     4141 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4201 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4261 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4321 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4381 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4441 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4501 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4561 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     4621 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     4681 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     4741 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     4801 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     4861 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     4921 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     4981 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     5041 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     5101 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     5161 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     5221 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     5281 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     5341 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     5401 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     5461 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     5521 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     5581 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5641 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     5701 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     5761 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     5821 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     5881 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     5941 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     6001 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     6061 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     6121 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     6181 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     6241 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     6301 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     6361 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     6421 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     6481 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     6541 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     6601 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     6661 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     6721 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     6781 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     6841 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     6901 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     6961 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     7021 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
     7081 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca t
//
