LOCUS       sequence-479206-        6894 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  VidC-bla 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 6894)
  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 6894)
  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-21_GSS-6xHis-SUMO-VidC-bla-FLAG
FEATURES             Location/Qualifiers
     source          1..6894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             176..199
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(252..270)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      266..313
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      350..805
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(437..456)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     647..668
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        832..936
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(937..1005,1006..1797)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1155..1174)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1968..2556
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2457..2476
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2710..2727
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2742..2884
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2810..2829)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2970..2992
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2986..3177)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3986..5068)
                     /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     5030..5049
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5069..5146)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5352..5371
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5455..5474
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5455..5473
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5474..5498
                     /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             5513..5535
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5555..5572
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             5582..5599
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             5606..5899
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
ORIGIN
        1 catcgctgac aaatctggtg ctggtgaacg tggttctcgt ggtatcatcg ctgctctggg
       61 tccggacggt aaaccgtctc gtatcgttgt tatctacacc accggttctc aggctaccat
      121 ggacgaacgt aaccgtcaga tcgctgaaat cggtgcttct ctgatcaaac actgggacta
      181 caaggacgac gacgacaagt aagatccggc tgctaacaaa gcccgaaagg aagctgagtt
      241 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
      301 gaggggtttt ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt
      361 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
      421 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
      481 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
      541 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga
      601 tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc
      661 caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg
      721 ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt
      781 aacaaaatat taacgtttac aatttcaggt ggcacttttc ggggaaatgt gcgcggaacc
      841 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
      901 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
      961 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     1021 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     1081 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     1141 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     1201 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     1261 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     1321 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     1381 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     1441 gaagccatac caaacgacga gcgtgacacc acgatgcctg cagcaatggc aacaacgttg
     1501 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     1561 atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt
     1621 attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg
     1681 ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg
     1741 gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg
     1801 tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     1861 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     1921 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     1981 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     2041 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     2101 ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     2161 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     2221 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     2281 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     2341 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     2401 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     2461 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     2521 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     2581 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     2641 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
     2701 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc
     2761 cttacgcatc tgtgcggtat ttcacaccgc atatatggtg cactctcagt acaatctgct
     2821 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     2881 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     2941 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     3001 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     3061 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     3121 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     3181 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     3241 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     3301 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     3361 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     3421 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     3481 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     3541 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     3601 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggggccg ccatgccggc
     3661 gataatggcc tgcttctcgc cgaaacgttt ggtggcggga ccagtgacga aggcttgagc
     3721 gagggcgtgc aagattccga ataccgcaag cgacaggccg atcatcgtcg cgctccagcg
     3781 aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc acctgtccta cgagttgcat
     3841 gataaagaag acagtcataa gtgcggcgac gatagtcatg ccccgcgccc accggaagga
     3901 gctgactggg ttgaaggctc tcaagggcat cggtcgagat cccggtgcct aatgagtgag
     3961 ctaacttaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     4021 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgcca
     4081 gggtggtttt tcttttcacc agtgagacgg gcaacagctg attgcccttc accgcctggc
     4141 cctgagagag ttgcagcaag cggtccacgc tggtttgccc cagcaggcga aaatcctgtt
     4201 tgatggtggt taacggcggg atataacatg agctgtcttc ggtatcgtcg tatcccacta
     4261 ccgagatatc cgcaccaacg cgcagcccgg actcggtaat ggcgcgcatt gcgcccagcg
     4321 ccatctgatc gttggcaacc agcatcgcag tgggaacgat gccctcattc agcatttgca
     4381 tggtttgttg aaaaccggac atggcactcc agtcgccttc ccgttccgct atcggctgaa
     4441 tttgattgcg agtgagatat ttatgccagc cagccagacg cagacgcgcc gagacagaac
     4501 ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa tgcgaccaga tgctccacgc
     4561 ccagtcgcgt accgtcttca tgggagaaaa taatactgtt gatgggtgtc tggtcagaga
     4621 catcaagaaa taacgccgga acattagtgc aggcagcttc cacagcaatg gcatcctggt
     4681 catccagcgg atagttaatg atcagcccac tgacgcgttg cgcgagaaga ttgtgcaccg
     4741 ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga caccaccacg ctggcaccca
     4801 gttgatcggc gcgagattta atcgccgcga caatttgcga cggcgcgtgc agggccagac
     4861 tggaggtggc aacgccaatc agcaacgact gtttgcccgc cagttgttgt gccacgcggt
     4921 tgggaatgta attcagctcc gccatcgccg cttccacttt ttcccgcgtt ttcgcagaaa
     4981 cgtggctggc ctggttcacc acgcgggaaa cggtctgata agagacaccg gcatactctg
     5041 cgacatcgta taacgttact ggtttcacat tcaccaccct gaattgactc tcttccgggc
     5101 gctatcatgc cataccgcga aaggttttgc gccattcgat ggtgtccggg atctcgacgc
     5161 tctcccttat gcgactcctg cattaggaag cagcccagta gtaggttgag gccgttgagc
     5221 accgccgccg caaggaatgg tgcatgcaag gagatggcgc ccaacagtcc cccggccacg
     5281 gggcctgcca ccatacccac gccgaaacaa gcgctcatga gcccgaagtg gcgagcccga
     5341 tcttccccat cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg
     5401 atgccggcca cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac
     5461 gactcactat aggggaattg tgagcggata acaattcccc tctagaaata attttgttta
     5521 actttaagaa ggagatatac atatgggcag cagccatcat catcatcatc acggcagcgg
     5581 cctggtgccg cgcggcagcg ctagcatgtc ggactcagaa gtcaatcaag aagctaagcc
     5641 agaggtcaag ccagaagtca agcctgagac tcacatcaat ttaaaggtgt ccgatggatc
     5701 ttcagagatc ttcttcaaga tcaaaaagac cactccttta agaaggctga tggaagcgtt
     5761 cgctaaaaga cagggtaagg aaatggactc cttaagattc ttgtacgacg gtattagaat
     5821 tcaagctgat cagacccctg aagatttgga catggaggat aacgatatta ttgaggctca
     5881 cagagaacag attggtggta tgattgagga gaaaaaggtc accgtgcagg agctgagaga
     5941 attgtatctg tctggagagt acacgatcga aatagatacc cccgacggat accagaccat
     6001 tgggaaatgg tttgataagg gcgtcttatc aatggtacgg gtcgcgactg ctacatacga
     6061 gacagtttgc gcattcaacc atatgatcca actcgccgac aatacctggg tgcaagcatg
     6121 cgaactggac gtaggtgttg atattcagac agccgccggg atccagccag tgatgcttgt
     6181 ggaggacact agtgatgctg aatgttatga tttcgaagtg atgcacccta atcacaggta
     6241 ctatggcgac ggtatcgtga gccataactc cggcaagatg cacccggaaa ccctggttaa
     6301 agttaaagac gctgaagacc agctgggtgc tcgtgttggt tacatcgaac tggacctgaa
     6361 ctctggtaaa atcctggaat ctttccgtcc ggaagaacgt ttcccgatga tgtctacctt
     6421 caaagttctg ctgtgcggtg ctgttctgtc tcgtgttgac gctggtcagg aacagctggg
     6481 tcgtcgtatc cactactctc agaacgacct ggttgaatac tctccggtta ccgaaaaaca
     6541 cctgaccgac ggtatgaccg ttcgtgaact gtgctctgct gctatcacca tgtctgacaa
     6601 caccgctgct aacctgctgc tgaccaccat cggtggtccg aaagaactga ccgctttcct
     6661 gcacaacatg ggtgaccacg ttacccgtct ggaccgttgg gaaccggaac tgaacgaagc
     6721 tatcccgaac gacgaacgtg acaccaccat gccggctgct atggctacca ccctgcgtaa
     6781 actgctgacc ggtgaactgc tgaccctggc ttctcgtcag cagctgatcg actggatgga
     6841 agctgacaaa gttgctggtc cgctgctgcg ttctgctctg ccggctggtt ggtt
//
