LOCUS       sequence-479203-        6260 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  VidC-EGF 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 6260)
  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 6260)
  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-EGF-FLAG
FEATURES             Location/Qualifiers
     source          1..6260
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      72..660
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     561..580
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     814..831
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    846..985
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(911..930)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1071..1093
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1087..1278)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(2087..3169)
                     /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     3131..3150
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3170..3247)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     3453..3472
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     3556..3575
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3556..3574
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    3575..3599
                     /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             3614..3636
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             3656..3673
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3683..3700
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             3707..4000
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             4541..4564
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(4617..4635)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4631..4678
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      4715..5170
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4802..4821)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5012..5033
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5196..5300
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5301..5369,5370..6161)
                     /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(5519..5538)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
       61 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
      121 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
      181 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
      241 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
      301 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
      361 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
      421 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
      481 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
      541 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
      601 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
      661 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
      721 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
      781 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
      841 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
      901 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
      961 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     1021 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     1081 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     1141 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     1201 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     1261 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     1321 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     1381 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     1441 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     1501 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     1561 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     1621 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     1681 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     1741 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     1801 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     1861 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     1921 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     1981 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     2041 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     2101 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     2161 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     2221 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     2281 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     2341 cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa
     2401 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     2461 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     2521 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     2581 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     2641 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     2701 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     2761 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     2821 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     2881 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     2941 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     3001 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     3061 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     3121 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     3181 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     3241 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     3301 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     3361 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     3421 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     3481 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     3541 tcgatcccgc gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc
     3601 ctctagaaat aattttgttt aactttaaga aggagatata catatgggca gcagccatca
     3661 tcatcatcat cacggcagcg gcctggtgcc gcgcggcagc gctagcatgt cggactcaga
     3721 agtcaatcaa gaagctaagc cagaggtcaa gccagaagtc aagcctgaga ctcacatcaa
     3781 tttaaaggtg tccgatggat cttcagagat cttcttcaag atcaaaaaga ccactccttt
     3841 aagaaggctg atggaagcgt tcgctaaaag acagggtaag gaaatggact ccttaagatt
     3901 cttgtacgac ggtattagaa ttcaagctga tcagacccct gaagatttgg acatggagga
     3961 taacgatatt attgaggctc acagagaaca gattggtggt atgattgagg agaaaaaggt
     4021 caccgtgcag gagctgagag aattgtatct gtctggagag tacacgatcg aaatagatac
     4081 ccccgacgga taccagacca ttgggaaatg gtttgataag ggcgtcttat caatggtacg
     4141 ggtcgcgact gctacatacg agacagtttg cgcattcaac catatgatcc aactcgccga
     4201 caatacctgg gtgcaagcat gcgaactgga cgtaggtgtt gatattcaga cagccgccgg
     4261 gatccagcca gtgatgcttg tggaggacac tagtgatgct gaatgttatg atttcgaagt
     4321 gatgcaccct aatcacaggt actatggcga cggtatcgtg agccataact ccggcaagat
     4381 gaacagtgat tcggaatgtc cgttgagcca tgatggttat tgcctgcacg acggggtttg
     4441 catgtacatc gaagcgctgg acaaatacgc ctgtaattgc gtcgtgggct atattggcga
     4501 acggtgtcag tatcgtgatt taaaatggtg ggagcttcgc gactacaagg acgacgacga
     4561 caagtaagat ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc
     4621 tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     4681 gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc
     4741 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     4801 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     4861 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     4921 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc
     4981 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca
     5041 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat
     5101 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg
     5161 cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     5221 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     5281 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     5341 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     5401 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     5461 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     5521 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     5581 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     5641 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     5701 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     5761 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     5821 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     5881 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     5941 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6001 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6061 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6121 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6181 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     6241 tcctttttga taatctcatg
//
