LOCUS       sequence-137259-        6154 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of monomeric enhanced green fluorescent protein
            (mEGFP) containing SnoopTag at the N-terminus and SpyTag at the 
            C-terminus, for programmed polyprotein synthesis..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6154)
  AUTHORS   Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, 
            Howarth M
  TITLE     Programmable polyproteams built using twin peptide superglues.
  JOURNAL   Proc Natl Acad Sci U S A. 2016 Jan 19. pii: 201519214.
  PUBMED    26787909
REFERENCE   2  (bases 1 to 6154)
  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: "Proc Natl 
            Acad Sci U S A. 2016 Jan 19. pii: 201519214."
COMMENT     Sequence Label: pET28a SnoopTag-mEGFP-SpyTag
FEATURES             Location/Qualifiers
     source          1..6154
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(170..208)
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     CDS             complement(join(245..955,956..958))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(287..308)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     636..655
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     895..916
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(983..1018)
                     /label=SnoopTag
     CDS             complement(1028..1045)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1055..1072)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1091..1113
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1128..1152
                     /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)."
     primer_bind     complement(1152..1171)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1153..1171)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1255..1274)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1480..1557
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1558..2640
                     /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     complement(1577..1596)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3449..3640
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3634..3656)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3742..3884
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3797..3816
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3899..3916)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4070..4658)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4150..4169)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4769..4788)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4780..5595
                     /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             4780..5595
                     /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     complement(4853..4872)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5688..6143)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5825..5846)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6037..6056
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagttattac ttcgtcggct
      181 tgtaggcgtc caccatcacg atgtgggcgg aaccgctccc gctaccggaa ccttccccgg
      241 atcccttgta cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca
      301 ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag cttggactgg gtgctcaggt
      361 agtggttgtc gggcagcagc acggggccgt cgccgatggg ggtgttctgc tggtagtggt
      421 cggcgagctg cacgctgccg tcctcgatgt tgtggcggat cttgaagttc accttgatgc
      481 cgttcttctg cttgtcggcc atgatataga cgttgtggct gttgtagttg tactccagct
      541 tgtgccccag gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca
      601 ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga
      661 agaagatggt gcgctcctgg acgtagcctt cgggcatggc ggacttgaag aagtcgtgct
      721 gcttcatgtg gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga
      781 gggtgggcca gggcacgggc agcttgccgg tggtgcagat gaacttcagg gtcagcttgc
      841 cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc
      901 cgtccagctc gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc ttgctcactc
      961 cggatcccga ctcaccactg cctttgttca ctttaataaa ttcaatatcg cccagtttcc
     1021 ccatatggct gccgcgcggc accaggccgc tgctgtgatg atgatgatga tggctgctgc
     1081 ccatggtata tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc
     1141 gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct
     1201 acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata
     1261 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt
     1321 tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc
     1381 atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc
     1441 taatgcagga gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa
     1501 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg
     1561 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc
     1621 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg
     1681 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg
     1741 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg
     1801 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga
     1861 agcggcgtcg aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg
     1921 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact
     1981 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc
     2041 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa
     2101 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg
     2161 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt
     2221 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg
     2281 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg
     2341 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt
     2401 tatatcccgc cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg
     2461 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc
     2521 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg
     2581 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     2641 gcgcaacgca attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc
     2701 cttgagagcc ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc
     2761 cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg
     2821 ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc
     2881 ggtattcgga atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg
     2941 tttcggcgag aagcaggcca ttatcgccgg catggcggcc ccacgggtgc gcatgatcgt
     3001 gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc cttactggtt agcagaatga
     3061 atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc aaaacgtctg cgacctgagc
     3121 aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt ctggaaacgc ggaagtcagc
     3181 gccctgcacc attatgttcc ggatctgcat cgcaggatgc tgctggctac cctgtggaac
     3241 acctacatct gtattaacga agcgctggca ttgaccctga gtgatttttc tctggtcccg
     3301 ccgcatccat accgccagtt gtttaccctc acaacgttcc agtaaccggg catgttcatc
     3361 atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc ggtatcatta cccccatgaa
     3421 cagaaatccc ccttacacgg aggcatcagt gaccaaacag gaaaaaaccg cccttaacat
     3481 ggcccgcttt atcagaagcc agacattaac gcttctggag aaactcaacg agctggacgc
     3541 ggatgaacag gcagacatct gtgaatcgct tcacgaccac gctgatgagc tttaccgcag
     3601 ctgcctcgcg cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac
     3661 ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc
     3721 gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata gcggagtgta
     3781 tactggctta actatgcggc atcagagcag attgtactga gagtgcacca tatatgcggt
     3841 gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct
     3901 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3961 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4021 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4081 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4141 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4201 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4261 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4321 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4381 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4441 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4501 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4561 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4621 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4681 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata
     4741 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     4801 acgtcttgct ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     4861 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     4921 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     4981 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     5041 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     5101 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     5161 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     5221 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     5281 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca
     5341 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     5401 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     5461 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     5521 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     5581 gatgagtttt tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa
     5641 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgaaa ttgtaaacgt
     5701 taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt ttaaccaata
     5761 ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag ggttgagtgt
     5821 tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg tcaaagggcg
     5881 aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat caagtttttt
     5941 ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc gatttagagc
     6001 ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga aaggagcggg
     6061 cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac ccgccgcgct
     6121 taatgcgccg ctacagggcg cgtcccattc gcca
//
