LOCUS       sequence-347993-        6771 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 6771)
  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 6771)
  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: GDAP2-c005
FEATURES             Location/Qualifiers
     source          1..6771
                     /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             1548..1565
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1618..1636)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1632..1679
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1716..2171
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1803..1822)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2013..2034
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2263..3078)
                     /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(2263..3078)
                     /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     2986..3005
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3070..3089
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3200..3788
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3689..3708
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3942..3959
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3974..4113
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4039..4058)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4199..4221
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4215..4406)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5215..6297)
                     /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     6259..6278
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6298..6375)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6581..6600
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6684..6703
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6684..6702
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6703..6727
                     /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             6742..6764
                     /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 atggatccct taggtgcacc ttcccagttt gtggatgtgg atacactacc aagctggggt
      841 gactcatgcc aagatgaatt aaattcctct gatactacag ctgaaatatt tcaggaagac
      901 actgttcgat caccttttct ttataataag gacgtcaatg gaaaagtggt tctttggaaa
      961 ggagatgtgg cattactgaa ctgtacagcc attgtgaata ccagcaatga aagtctcaca
     1021 gataagaatc ctgtgtcaga aagtatcttc atgcttgcag ggcctgattt gaaggaagat
     1081 ctccagaaac ttaaagggtg ccgaacaggt gaagcaaaat tgacaaaagg attcaatcta
     1141 gctgcccggt tcatcattca cacagtggga cctaaatata aaagccgcta tcgcacagca
     1201 gctgagagtt ccctttatag ctgctacaga aacgtacttc aactagcaaa agagcagtca
     1261 atgtcttctg ttggcttctg tgtcatcaat tctgcaaaac gtggttatcc tttagaggat
     1321 gcaacacaca tagcacttcg cactgtaaga agattcctag agattcatgg ggaaaccatt
     1381 gaaaaagtag tatttgctgt ctctgatctt gaagagggta cttaccaaaa gctgctacct
     1441 ctctacttcc caaggtcatt aaaagaggag aattaagacg gtctccagta aaggtggata
     1501 cggatccgaa ttcgagctcc gtcgacaagc ttgcggccgc actcgagcac caccaccacc
     1561 accactgaga tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg
     1621 ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc
     1681 tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag
     1741 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     1801 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     1861 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     1921 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     1981 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     2041 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     2101 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     2161 gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt
     2221 ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa actcatcgag
     2281 catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt tttgaaaaag
     2341 ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg caagatcctg
     2401 gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc
     2461 aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg
     2521 caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc
     2581 aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa
     2641 tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa
     2701 cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa
     2761 tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa
     2821 atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc
     2881 tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg
     2941 cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt
     3001 atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc aagacgtttc
     3061 ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag acagttttat
     3121 tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3181 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3241 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3301 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3361 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3421 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3481 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3541 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3601 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3661 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     3721 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3781 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     3841 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     3901 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     3961 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4021 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4081 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4141 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4201 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     4261 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     4321 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     4381 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     4441 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     4501 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     4561 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     4621 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     4681 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     4741 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     4801 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     4861 atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     4921 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     4981 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     5041 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     5101 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     5161 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     5221 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     5281 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     5341 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     5401 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     5461 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     5521 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     5581 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     5641 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     5701 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     5761 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     5821 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     5881 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     5941 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     6001 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     6061 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     6121 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     6181 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     6241 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     6301 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     6361 ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc
     6421 agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg
     6481 agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag
     6541 cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg
     6601 cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga
     6661 tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
     6721 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca t
//
