LOCUS       sequence-479245-        6747 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  VidC-EGFP construct that can be purified via His-tag and undergo 
            N-terminal conjugation using the VEPL system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6747)
  AUTHORS   Gharios R, Li A, Kopyeva I, Francis RM, DeForest CA
  TITLE     One-Step Purification and N-Terminal Functionalization of Bioactive 
            Proteins via Atypically Split Inteins.
  JOURNAL   Bioconjug Chem. 2024 Jun 19;35(6):750-757. doi: 
            10.1021/acs.bioconjchem.4c00223. Epub 2024 May 30.
  PUBMED    38815180
REFERENCE   2  (bases 1 to 6747)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acs.bioconjchem.4c00223"; journalName: "Bioconjug Chem"; 
            date: "2024-06-19- 19"; volume: "35"; issue: "6"; pages: "750-757"
COMMENT     Sequence Label: pET-29_GSS-6xHis-SUMO-VidC-EGFP-FLAG
FEATURES             Location/Qualifiers
     source          1..6747
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1637..1828
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1822..1844)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1930..2069
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1985..2004
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2084..2101)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2255..2843)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2335..2354)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2954..2973)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2965..3780
                     /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             2965..3780
                     /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(3038..3057)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3872..4327)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4009..4030)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4221..4240
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(4364..4411)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     4407..4425
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(4478..4495)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(4539..4562)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(join(4563..5273,5274..5276,5277..5279))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(4563..5273,5274..5276,5277..5279))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4605..4626)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     4954..4973
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5213..5234
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(5658..5951)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(5958..5975)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(5985..6002)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             6022..6044
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    6059..6083
                     /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(6083..6102)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6084..6102)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6190..6209)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6415..6492
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(6493..6747,1..828)
                     /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(6512..6531)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa
       61 gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac
      121 tatccgctgg atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg
      181 ttatttcttg atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac
      241 ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta
      301 gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc
      361 actcgcaatc aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt
      421 tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc
      481 aacgatcaga tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt
      541 gcggacatct cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg
      601 ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg
      661 caactctctc agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa
      721 agaaaaacca ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
      781 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
      841 taatgtaagt tagctcactc attaggcacc gggatctcga ccgatgccct tgagagcctt
      901 caacccagtc agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac
      961 tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg
     1021 cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat
     1081 cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa
     1141 gcaggccatt atcgccggca tggcggcccc acgggtgcgc atgatcgtgc tcctgtcgtt
     1201 gaggacccgg ctaggctggc ggggttgcct tactggttag cagaatgaat caccgatacg
     1261 cgagcgaacg tgaagcgact gctgctgcaa aacgtctgcg acctgagcaa caacatgaat
     1321 ggtcttcggt ttccgtgttt cgtaaagtct ggaaacgcgg aagtcagcgc cctgcaccat
     1381 tatgttccgg atctgcatcg caggatgctg ctggctaccc tgtggaacac ctacatctgt
     1441 attaacgaag cgctggcatt gaccctgagt gatttttctc tggtcccgcc gcatccatac
     1501 cgccagttgt ttaccctcac aacgttccag taaccgggca tgttcatcat cagtaacccg
     1561 tatcgtgagc atcctctctc gtttcatcgg tatcattacc cccatgaaca gaaatccccc
     1621 ttacacggag gcatcagtga ccaaacagga aaaaaccgcc cttaacatgg cccgctttat
     1681 cagaagccag acattaacgc ttctggagaa actcaacgag ctggacgcgg atgaacaggc
     1741 agacatctgt gaatcgcttc acgaccacgc tgatgagctt taccgcagct gcctcgcgcg
     1801 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     1861 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     1921 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtata ctggcttaac
     1981 tatgcggcat cagagcagat tgtactgaga gtgcaccatt gcggtgtgaa ataccgcaca
     2041 gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc ttcctcgctc actgactcgc
     2101 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2161 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2221 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2281 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2341 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2401 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2461 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2521 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2581 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2641 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag
     2701 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2761 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2821 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     2881 agtggaacga aaactcacgt taagggattt tggtcatgaa caataaaact gtctgcttac
     2941 ataaacagta atacaagggg tgttatgagc catattcaac gggaaacgtc ttgctctagg
     3001 ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat
     3061 gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg
     3121 tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcagacta
     3181 aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat
     3241 gatgcatggt tactcaccac tgcgatcccc gggaaaacag cattccaggt attagaagaa
     3301 tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat
     3361 tcgattcctg tttgtaattg tccttttaac agcgatcgcg tatttcgtct cgctcaggcg
     3421 caatcacgaa tgaataacgg tttggttgat gcgagtgatt ttgatgacga gcgtaatggc
     3481 tggcctgttg aacaagtctg gaaagaaatg cataaacttt tgccattctc accggattca
     3541 gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata
     3601 ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta
     3661 tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt
     3721 attgataatc ctgatatgaa taaattgcag tttcatttga tgctcgatga gtttttctaa
     3781 gaattaattc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     3841 tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa
     3901 ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa
     3961 atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac
     4021 aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag
     4081 ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttgaggtc gaggtgccgt
     4141 aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg
     4201 gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca
     4261 agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag
     4321 ggcgcgtccc attcgccaat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga
     4381 cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact
     4441 cagcttcctt tcgggctttg ttagcagccg gatctcagtg gtggtggtgg tggtgctcga
     4501 gtgcggccgc aagcttgtcg acggagctcg aattcttact tgtcgtcgtc gtccttgtag
     4561 tccttgtaca gctcgtccat gccgagagtg atcccggcgg cggtcacgaa ctccagcagg
     4621 accatgtgat cgcgcttctc gttggggtct ttgctcaggg cggactgggt gctcaggtag
     4681 tggttgtcgg gcagcagcac ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg
     4741 gcgagctgca cgctgccgtc ctcgatgttg tggcggatct tgaagttcac cttgatgccg
     4801 ttcttctgct tgtcggccat gatatagacg ttgtggctgt tgtagttgta ctccagcttg
     4861 tgccccagga tgttgccgtc ctccttgaag tcgatgccct tcagctcgat gcggttcacc
     4921 agggtgtcgc cctcgaactt cacctcggcg cgggtcttgt agttgccgtc gtccttgaag
     4981 aagatggtgc gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc
     5041 ttcatgtggt cggggtagcg gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg
     5101 gtgggccagg gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg
     5161 taggtggcat cgccctcgcc ctcgccggac acgctgaact tgtggccgtt tacgtcgccg
     5221 tccagctcga ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatc
     5281 ttgccggagt tatggctcac gataccgtcg ccatagtacc tgtgattagg gtgcatcact
     5341 tcgaaatcat aacattcagc atcactagtg tcctccacaa gcatcactgg ctggatcccg
     5401 gcggctgtct gaatatcaac acctacgtcc agttcgcatg cttgcaccca ggtattgtcg
     5461 gcgagttgga tcatatggtt gaatgcgcaa actgtctcgt atgtagcagt cgcgacccgt
     5521 accattgata agacgccctt atcaaaccat ttcccaatgg tctggtatcc gtcgggggta
     5581 tctatttcga tcgtgtactc tccagacaga tacaattctc tcagctcctg cacggtgacc
     5641 tttttctcct caatcatacc accaatctgt tctctgtgag cctcaataat atcgttatcc
     5701 tccatgtcca aatcttcagg ggtctgatca gcttgaattc taataccgtc gtacaagaat
     5761 cttaaggagt ccatttcctt accctgtctt ttagcgaacg cttccatcag ccttcttaaa
     5821 ggagtggtct ttttgatctt gaagaagatc tctgaagatc catcggacac ctttaaattg
     5881 atgtgagtct caggcttgac ttctggcttg acctctggct tagcttcttg attgacttct
     5941 gagtccgaca tgctagcgct gccgcgcggc accaggccgc tgccgtgatg atgatgatga
     6001 tggctgctgc ccatatgtat atctccttct taaagttaaa caaaattatt tctagagggg
     6061 aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat
     6121 cgatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc acaggtgcgg
     6181 ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc cacttcgggc
     6241 tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg ggactgttgg
     6301 gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc ctcaacctac
     6361 tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat cccggacacc
     6421 atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga gagtcaattc
     6481 agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc cggtgtctct
     6541 tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa aacgcgggaa
     6601 aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc acaacaactg
     6661 gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct gcacgcgccg
     6721 tcgcaaattg tcgcggcgat taaatct
//
