LOCUS       sequence-322297-        6671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP fusion of a multisite mutant of the Af1521 protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6671)
  AUTHORS   Garcia-Saura AG, Herzog LK, Dantuma NP, Schuler H
  TITLE     MacroGreen, a simple tool for detection of ADP-ribosylated proteins.
  JOURNAL   Commun Biol. 2021 Jul 28;4(1):919. doi: 10.1038/s42003-021-02439-w.
  PUBMED    34321589
REFERENCE   2  (bases 1 to 6671)
  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: "Commun 
            Biol."; date: "2021-07-28"; volume: "4(1):919. doi"; pages: " 
            10.1038/s42003-021-02439-w"
COMMENT     Sequence Label: pNIC28-MacroGreen
FEATURES             Location/Qualifiers
     source          1..6671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..18
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             43..63
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQS"
     CDS             join(676..678,679..681,682..1395)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(676..678,679..681,682..1392)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(721..742)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(982..1001)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1329..1350
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1445..1462
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1515..1533)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1529..1576
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1613..2068
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1700..1719)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1910..1931
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(2160..2975)
                     /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(2160..2975)
                     /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     2883..2902
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2967..2986
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3097..3685
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3586..3605
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3839..3856
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3871..4010
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3936..3955)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4096..4118
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4112..4303)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5112..6194)
                     /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
                     EACKAAVHNLLAQRVSGLIINYQLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     6156..6175
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6195..6272)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6478..6497
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6581..6600
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6581..6599
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6600..6624
                     /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             6639..6661
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 caccatcatc atcatcattc ttctggtgta gatctgggta ccgagaacct gtacttccaa
       61 tccatggagg ttctgtttga agccaaagtt ggtgatataa ccctgaaact ggcacagggt
      121 gatattaccc agtatccggc aaaagcaatt gttaatgcag caaatgaacg tctggaacat
      181 ggtggtgaag ttgcatatgc cattgcaaaa gcatgtgccg gtgatgcagg tctgtatacc
      241 gaaatttcaa aaaaagcaat gcgcgaacag ttcggtcgcg attatattga tcatggtgaa
      301 gtggttgtta cccctgcaat gaatctggaa gaacgtggta tcaaatatgt gtttcatacc
      361 gttggtccga tttgtagcgg tatgtggtca gaagaactga aagagaaact gtataaagca
      421 tttctgggtc cgctggaaaa agccgaagaa atgggtgttg aaagcattgc atttccggca
      481 gttagcgcag gtcgtcgtgg ttgtgattta gaaaaagttg ttgaaacctt tctggaagcc
      541 gtgaaaaact ttaaaggtag cgcagttaaa gaagtggccc tggttattta tgatcgtaaa
      601 agcgcagaag ttgccctgaa agtttttgaa cgtagcctga gcagcaaagg tggttatggt
      661 ctgaatgata ctagtatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
      721 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
      781 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
      841 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
      901 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
      961 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     1021 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     1081 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     1141 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     1201 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     1261 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     1321 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     1381 gagctgtaca agtaatgagg atccgaattc gagctccgtc gacaagcttg cggccgcact
     1441 cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga
     1501 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1561 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc
     1621 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     1681 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     1741 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     1801 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     1861 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     1921 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     1981 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2041 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     2101 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg aattaattct
     2161 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     2221 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
     2281 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
     2341 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact
     2401 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc aacaggccag
     2461 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
     2521 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa
     2581 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
     2641 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
     2701 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt
     2761 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
     2821 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
     2881 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc
     2941 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
     3001 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3061 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3121 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3181 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3241 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3301 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3361 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     3421 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     3481 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     3541 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     3601 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     3661 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     3721 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     3781 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     3841 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     3901 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3961 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg ccccgacacc
     4021 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac
     4081 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac
     4141 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca cagatgtctg
     4201 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc tggcttctga
     4261 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc tccgtgtaag
     4321 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg ctcacgatac
     4381 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa caactggcgg
     4441 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc ttcgttaata
     4501 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc cggaacataa
     4561 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa ccgaagacca
     4621 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc
     4681 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc cgggtcctca
     4741 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata atggcctgct
     4801 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga
     4861 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc
     4921 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag
     4981 tcataagtgc ggcgacgata gtcatgtccc gcgcccaccg gaaggagctg actgggttga
     5041 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa
     5101 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5161 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt
     5221 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc
     5281 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac
     5341 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatatccgca
     5401 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg
     5461 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa
     5521 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg
     5581 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct
     5641 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg
     5701 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac
     5761 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagctgatag
     5821 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct
     5881 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga
     5941 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg
     6001 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc
     6061 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg
     6121 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac
     6181 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgccata
     6241 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc ccttatgcga
     6301 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg ccgccgcaag
     6361 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     6421 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     6481 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     6541 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact cactataggg
     6601 gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt taagaaggag
     6661 atatacatat g
//
