LOCUS       sequence-383674-        5027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PI-SceI (VDE) homing endonuclease/intein from the T7 
            promoter that contains the D218A active site mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5027)
  AUTHORS   Gimble FS, Stephens BW
  TITLE     Substitutions in conserved dodecapeptide motifs that uncouple the 
            DNA binding and DNA cleavage activities of PI-SceI endonuclease.
  JOURNAL   J Biol Chem. 1995 Mar 17;270(11):5849-56. doi: 
            10.1074/jbc.270.11.5849.
  PUBMED    7890714
REFERENCE   2  (bases 1 to 5027)
  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"; doi: 
            "10.1074/jbc.270.11"; journalName: "J Biol Chem"; date: "1995-03-17-
            17"; volume: "270"; issue: "11"; pages: "5849-56"
COMMENT     Sequence Label: pET23PI-Sce ESARC D218A
FEATURES             Location/Qualifiers
     source          1..5027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     213..232
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        213..231
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             263..285
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             296..1114
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae VMA1"
                     /product="5' region of the intein from the yeast Vma1 
                     subunit of the vacuolar ATPase (Chong et al., 1998)"
                     /label=Sce VMA intein 5' region
                     /translation="CFAKGTNVLMADGSIECIENIEVGNKVMGKDGRPREVIKLPRGRE
                     TMYSVVQKSQHRAHKSDSSREVPELLKFTCNATHELVVRTPRSVRRLSRTIKGVEYFEV
                     ITFEMGQKKAPDGRIVELVKEVSKSYPISEGPERANELVESYRKASNKAYFEWTIEARD
                     LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLLGLWIGAGLSD
                     RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRG"
     CDS             1570..1581
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1661..1690
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="ASMTGGQQMG"
     CDS             1740..1757
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1810..1828)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1824..1871
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1908..2363
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1995..2014)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2205..2226
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2389..2493
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2494..2562,2563..3354)
                     /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(2712..2731)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3525..4113
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4014..4033
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4267..4284
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4299..4438
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4364..4383)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4524..4546
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4540..4731)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc
       61 agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta aggcaacccc
      121 gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt ggccaggacc
      181 caacgctgcc cgagatctcg atcccgcgaa attaatacga ctcactatag ggagaccaca
      241 acggtttccc tctagaaata attttgttta actttaagaa ggagatatac atatgtgctt
      301 tgccaagggt accaatgttt taatggcgga tgggtctatt gaatgtattg aaaacattga
      361 ggttggtaat aaggtcatgg gtaaagatgg cagacctcgt gaggtaatta aattgcccag
      421 aggaagagaa actatgtaca gcgtcgtgca gaaaagtcag cacagagccc acaaaagtga
      481 ctcaagtcgt gaagtgccag aattactcaa gtttacgtgt aatgcgaccc atgagttggt
      541 tgttagaaca cctcgtagtg tccgccgttt gtctcgtacc attaagggtg tcgaatattt
      601 tgaagttatt acttttgaga tgggccaaaa gaaagccccc gacggtagaa ttgttgagct
      661 tgtcaaggaa gtttcaaaga gctacccaat atctgagggg cctgagagag ccaacgaatt
      721 agtagaatcc tatagaaagg cttcaaataa agcttatttt gagtggacta ttgaggccag
      781 agatctttct ctgttgggtt cccatgttcg taaagctacc taccagactt acgctccaat
      841 tctttatgag aatgaccact ttttcgacta catgcaaaaa agtaagtttc atctcaccat
      901 tgaaggtcca aaagtacttg cttatctcct aggcctatgg attggtgctg gattgtcgga
      961 ccgggcaact ttttcggttg attccagaga tacttctttg atggaacgtg ttactgaata
     1021 tgctgaaaag ttgaatttgt gcgccgagta taaggacaga aaagaaccac aagttgccaa
     1081 aactgttaat ttgtactcta aagttgtcag aggtaatggt attcgcaata atcttaatac
     1141 tgagaatcca ttatgggacg ctattgttgg cttaggattc ttgaaggacg gtgtcaaaaa
     1201 tattccttct ttcttgtcta cggacaatat cggtactcgt gaaacatttc tggccggtct
     1261 aattgattct gatggctacg taactgatga gcatggtatt aaagcaacaa taaagacaat
     1321 tcatacttct gtcagagatg gtttggtttc ccttgctcgt tctttaggct tagtagtctc
     1381 ggttaacgca gaacctgcta aggttgacat gaatggcacc aaacataaaa ttagttatgc
     1441 tatttatatg tctggtggag atgttttgct taacgttctt tcgaagtgtg ccggctctaa
     1501 aaaattcagg cctgctcccg ccgctgcttt tgcacgtgag tgccgcggat tttatttcga
     1561 gttacaagaa ttgaaggaag acgattatta tgggattact ttatctgatg attctgatca
     1621 tcagtttttg cttgccaacc aggttgtcgt ccataattag gctagcatga ctggtggaca
     1681 gcaaatgggt cgcggatccg aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc
     1741 accaccacca ccaccactga gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg
     1801 ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa cgggtcttga
     1861 ggggtttttt gctgaaagga ggaactatat ccggattggc gaatgggacg cgccctgtag
     1921 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     1981 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     2041 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     2101 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     2161 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     2221 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     2281 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     2341 caaaatatta acgcttacaa tttaggtggc acttttcggg gaaatgtgcg cggaacccct
     2401 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     2461 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     2521 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     2581 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     2641 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     2701 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     2761 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     2821 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     2881 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     2941 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3001 gccataccaa acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc
     3061 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3121 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3181 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     3241 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3301 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3361 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     3421 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     3481 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     3541 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     3601 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     3661 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     3721 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     3781 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     3841 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     3901 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     3961 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4021 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4081 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4141 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4201 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4261 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt
     4321 acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg
     4381 ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc
     4441 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     4501 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     4561 accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca
     4621 gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg
     4681 gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc
     4741 cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct
     4801 cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca
     4861 actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt
     4921 cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg
     4981 gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacg
//
