LOCUS       sequence-479244-        6681 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  VidC-mCherry 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 6681)
  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 6681)
  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-mCherry-FLAG
FEATURES             Location/Qualifiers
     source          1..6681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(63..80)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(234..822)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(314..333)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(933..952)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             944..1759
                     /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             944..1759
                     /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(1017..1036)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(1852..2307)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1989..2010)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2201..2220
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(2344..2391)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2387..2405
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(2458..2481)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(3568..3861)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(3868..3885)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(3895..3912)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             3932..3954
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    3969..3993
                     /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(3993..4012)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3994..4012)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4100..4119)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4325..4402
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4403..5485
                     /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(4422..4441)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             6294..6485
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(6479..6501)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    join(6587..6681,1..48)
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     6642..6661
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
       61 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
      121 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
      181 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
      241 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
      301 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
      361 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
      421 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
      481 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
      541 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
      601 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
      661 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
      721 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
      781 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
      841 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaac
      901 aataaaactg tctgcttaca taaacagtaa tacaaggggt gttatgagcc atattcaacg
      961 ggaaacgtct tgctctaggc cgcgattaaa ttccaacatg gatgctgatt tatatgggta
     1021 taaatgggct cgcgataatg tcgggcaatc aggtgcgaca atctatcgat tgtatgggaa
     1081 gcccgatgcg ccagagttgt ttctgaaaca tggcaaaggt agcgttgcca atgatgttac
     1141 agatgagatg gtcagactaa actggctgac ggaatttatg cctcttccga ccatcaagca
     1201 ttttatccgt actcctgatg atgcatggtt actcaccact gcgatccccg ggaaaacagc
     1261 attccaggta ttagaagaat atcctgattc aggtgaaaat attgttgatg cgctggcagt
     1321 gttcctgcgc cggttgcatt cgattcctgt ttgtaattgt ccttttaaca gcgatcgcgt
     1381 atttcgtctc gctcaggcgc aatcacgaat gaataacggt ttggttgatg cgagtgattt
     1441 tgatgacgag cgtaatggct ggcctgttga acaagtctgg aaagaaatgc ataaactttt
     1501 gccattctca ccggattcag tcgtcactca tggtgatttc tcacttgata accttatttt
     1561 tgacgagggg aaattaatag gttgtattga tgttggacga gtcggaatcg cagaccgata
     1621 ccaggatctt gccatcctat ggaactgcct cggtgagttt tctccttcat tacagaaacg
     1681 gctttttcaa aaatatggta ttgataatcc tgatatgaat aaattgcagt ttcatttgat
     1741 gctcgatgag tttttctaag aattaattca tgagcggata catatttgaa tgtatttaga
     1801 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gaaattgtaa
     1861 acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     1921 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga
     1981 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     2041 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt
     2101 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     2161 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     2221 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     2281 cgcttaatgc gccgctacag ggcgcgtccc attcgccaat ccggatatag ttcctccttt
     2341 cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta gttattgctc
     2401 agcggtggca gcagccaact cagcttcctt tcgggctttg ttagcagccg gatcttactt
     2461 gtcgtcgtcg tccttgtagt ccttgtacag ttcgtccata ccaccggtag agtgacggcc
     2521 ttccgcacgt tcgtactgtt caacgatggt gtagtcttcg ttgtgagagg tgatgtccag
     2581 cttgatatta acgttgtacg cacccggcag ttgaaccggc tttttcgctt tgtaggtggt
     2641 tttaacctcc gcgtcgtagt gaccgccgtc tttgagcttc agacgctgtt tgatttcgcc
     2701 tttcagcgca ccatcttccg ggtacatacg ttcagaagac gcttcccaac ccatggtttt
     2761 tttctgcata accggaccgt cagacgggaa gttggtgcca cgcagtttga ccttgtagat
     2821 gaactcgccg tcctgcagag aagagtcctg ggtaacggtg acaacaccac cgtcctcgaa
     2881 gttcataaca cgttcccatt tgaaaccttc agggaaagag agtttgaggt agtctggaat
     2941 gtccgccgga tgtttaacgt acgctttaga accgtacatg aactgcgggc tgaggatgtc
     3001 ccacgcgaac ggcagcggac cacctttggt aactttcagt ttcgcggtct gggtaccttc
     3061 gtacgggcga ccttcaccct caccttcaat ttcgaactca tgaccgttaa cagaaccttc
     3121 catgtgaact ttaaaacgca taaattcttt gatgatcgcc atgttgtctt cttcgccctt
     3181 ggaaaccatc ttgccggagt tatggctcac gataccgtcg ccatagtacc tgtgattagg
     3241 gtgcatcact tcgaaatcat aacattcagc atcactagtg tcctccacaa gcatcactgg
     3301 ctggatcccg gcggctgtct gaatatcaac acctacgtcc agttcgcatg cttgcaccca
     3361 ggtattgtcg gcgagttgga tcatatggtt gaatgcgcaa actgtctcgt atgtagcagt
     3421 cgcgacccgt accattgata agacgccctt atcaaaccat ttcccaatgg tctggtatcc
     3481 gtcgggggta tctatttcga tcgtgtactc tccagacaga tacaattctc tcagctcctg
     3541 cacggtgacc tttttctcct caatcatacc accaatctgt tctctgtgag cctcaataat
     3601 atcgttatcc tccatgtcca aatcttcagg ggtctgatca gcttgaattc taataccgtc
     3661 gtacaagaat cttaaggagt ccatttcctt accctgtctt ttagcgaacg cttccatcag
     3721 ccttcttaaa ggagtggtct ttttgatctt gaagaagatc tctgaagatc catcggacac
     3781 ctttaaattg atgtgagtct caggcttgac ttctggcttg acctctggct tagcttcttg
     3841 attgacttct gagtccgaca tgctagcgct gccgcgcggc accaggccgc tgccgtgatg
     3901 atgatgatga tggctgctgc ccatatgtat atctccttct taaagttaaa caaaattatt
     3961 tctagagggg aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg
     4021 ggatcgagat cgatctcgat cctctacgcc ggacgcatcg tggccggcat caccggcgcc
     4081 acaggtgcgg ttgctggcgc ctatatcgcc gacatcaccg atggggaaga tcgggctcgc
     4141 cacttcgggc tcatgagcgc ttgtttcggc gtgggtatgg tggcaggccc cgtggccggg
     4201 ggactgttgg gcgccatctc cttgcatgca ccattccttg cggcggcggt gctcaacggc
     4261 ctcaacctac tactgggctg cttcctaatg caggagtcgc ataagggaga gcgtcgagat
     4321 cccggacacc atcgaatggc gcaaaacctt tcgcggtatg gcatgatagc gcccggaaga
     4381 gagtcaattc agggtggtga atgtgaaacc agtaacgtta tacgatgtcg cagagtatgc
     4441 cggtgtctct tatcagaccg tttcccgcgt ggtgaaccag gccagccacg tttctgcgaa
     4501 aacgcgggaa aaagtggaag cggcgatggc ggagctgaat tacattccca accgcgtggc
     4561 acaacaactg gcgggcaaac agtcgttgct gattggcgtt gccacctcca gtctggccct
     4621 gcacgcgccg tcgcaaattg tcgcggcgat taaatctcgc gccgatcaac tgggtgccag
     4681 cgtggtggtg tcgatggtag aacgaagcgg cgtcgaagcc tgtaaagcgg cggtgcacaa
     4741 tcttctcgcg caacgcgtca gtgggctgat cattaactat ccgctggatg accaggatgc
     4801 cattgctgtg gaagctgcct gcactaatgt tccggcgtta tttcttgatg tctctgacca
     4861 gacacccatc aacagtatta ttttctccca tgaagacggt acgcgactgg gcgtggagca
     4921 tctggtcgca ttgggtcacc agcaaatcgc gctgttagcg ggcccattaa gttctgtctc
     4981 ggcgcgtctg cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat
     5041 agcggaacgg gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct
     5101 gaatgagggc atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc
     5161 aatgcgcgcc attaccgagt ccgggctgcg cgttggtgcg gacatctcgg tagtgggata
     5221 cgacgatacc gaagacagct catgttatat cccgccgtta accaccatca aacaggattt
     5281 tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt
     5341 gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa
     5401 tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt
     5461 ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt
     5521 aggcaccggg atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt
     5581 gggcgcgggg catgactatc gtcgccgcac ttatgactgt cttctttatc atgcaactcg
     5641 taggacaggt gccggcagcg ctctgggtca ttttcggcga ggaccgcttt cgctggagcg
     5701 cgacgatgat cggcctgtcg cttgcggtat tcggaatctt gcacgccctc gctcaagcct
     5761 tcgtcactgg tcccgccacc aaacgtttcg gcgagaagca ggccattatc gccggcatgg
     5821 cggccccacg ggtgcgcatg atcgtgctcc tgtcgttgag gacccggcta ggctggcggg
     5881 gttgccttac tggttagcag aatgaatcac cgatacgcga gcgaacgtga agcgactgct
     5941 gctgcaaaac gtctgcgacc tgagcaacaa catgaatggt cttcggtttc cgtgtttcgt
     6001 aaagtctgga aacgcggaag tcagcgccct gcaccattat gttccggatc tgcatcgcag
     6061 gatgctgctg gctaccctgt ggaacaccta catctgtatt aacgaagcgc tggcattgac
     6121 cctgagtgat ttttctctgg tcccgccgca tccataccgc cagttgttta ccctcacaac
     6181 gttccagtaa ccgggcatgt tcatcatcag taacccgtat cgtgagcatc ctctctcgtt
     6241 tcatcggtat cattaccccc atgaacagaa atccccctta cacggaggca tcagtgacca
     6301 aacaggaaaa aaccgccctt aacatggccc gctttatcag aagccagaca ttaacgcttc
     6361 tggagaaact caacgagctg gacgcggatg aacaggcaga catctgtgaa tcgcttcacg
     6421 accacgctga tgagctttac cgcagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
     6481 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     6541 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     6601 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     6661 actgagagtg caccatatat g
//
