LOCUS       sequence-383672-        3801 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PI-SceI (VDE) homing endonuclease/intein from the T7 
            promoter that contains the D326A active site mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3801)
  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 3801)
  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: pT7PI-Sce ESARC D326A
FEATURES             Location/Qualifiers
     source          1..3801
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             817..828
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             complement(join(1000..1791,1792..1860))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1623..1642
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1861..1965)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2033..2051
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      2180..2768
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2669..2688
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2922..2939
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     3174..3196
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3261..3280
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3261..3279
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             3311..3333
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(3344..3801,1..361)
                     /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
                     LSLLGSHVRKATYQTYAPILYENDHFFDYMQKSKFHLTIEGPKVLAYLLGLWIGDGLSD
                     RATFSVDSRDTSLMERVTEYAEKLNLCAEYKDRKEPQVAKTVNLYSKVVRG"
ORIGIN
        1 ttattttgag tggactattg aggccagaga tctttctctg ttgggttccc atgttcgtaa
       61 agctacctac cagacttacg ctccaattct ttatgagaat gaccactttt tcgactacat
      121 gcaaaaaagt aagtttcatc tcaccattga aggtccaaaa gtacttgctt atctcctagg
      181 tttatggatt ggtgatggat tgtcggaccg ggcaactttt tcggttgatt ccagagatac
      241 ttctttgatg gaacgtgtta ctgaatatgc tgaaaagttg aatttgtgcg ccgagtataa
      301 ggacagaaaa gaaccacaag ttgccaaaac tgttaatttg tactctaaag ttgtcagagg
      361 taatggtatt cgcaataatc ttaatactga gaatccatta tgggacgcta ttgttggctt
      421 aggattcttg aaggacggtg tcaaaaatat tccttctttc ttgtctacgg acaatatcgg
      481 tactcgtgaa acatttctgg ccggtctaat cgattctgcg ggctacgtaa ctgatgagca
      541 tggtattaaa gcaacaataa agacaattca tacttctgtc agagatggtt tggtttccct
      601 tgctcgttct ttaggcttag tagtctcggt taacgcagaa cctgctaagg ttgacatgaa
      661 tggcaccaaa cataaaatta gttatgctat ttatatgtct ggtggagatg ttttgcttaa
      721 cgttctttcg aagtgtgccg gctctaaaaa attcaggcct gctcccgccg ctgcttttgc
      781 acgtgagtgc cgcggatttt atttcgagtt acaagaattg aaggaagacg attattatgg
      841 gattacttta tctgatgatt ctgatcatca gtttttgctt gccaaccagg ttgtcgtcca
      901 taattaggct agcctgcagc ccaagcttat cgatgataag ctgtcaaaca tgagaattaa
      961 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     1021 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     1081 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     1141 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     1201 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     1261 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     1321 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     1381 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     1441 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     1501 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     1561 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaacacg
     1621 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     1681 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     1741 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     1801 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     1861 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     1921 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     1981 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     2041 tatcacgagg ccctttcgtc ttcaagaata aaaggatcta ggtgaagatc ctttttgata
     2101 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     2161 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     2221 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     2281 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     2341 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     2401 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     2461 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     2521 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     2581 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     2641 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     2701 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     2761 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     2821 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     2881 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     2941 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     3001 gcataggaag atcttccgga agatcttcct atggtgcact ctcagtacaa tctgctctga
     3061 tgcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     3121 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     3181 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggccca gcgattcgaa
     3241 cttctgatag acttcgaaat taatacgact cactataggg agaccacaac ggtttccctc
     3301 tagaaataat tttgtttaac tttaagaagg agatatacat atgtgctttg ccaagggtac
     3361 caatgtttta atggcggatg ggtctattga atgtattgaa aacattgagg ttggtaataa
     3421 ggtcatgggt aaagatggca gacctcgtga ggtaattaaa ttgcccagag gaagagaaac
     3481 tatgtacagc gtcgtgcaga aaagtcagca cagagcccac aaaagtgact caagtcgtga
     3541 agtgccagaa ttactcaagt ttacgtgtaa tgcgacccat gagttggttg ttagaacacc
     3601 tcgtagtgtc cgccgtttgt ctcgtaccat taagggtgtc gaatattttg aagttattac
     3661 ttttgagatg ggccaaaaga aagcccccga cggtagaatt gttgagcttg tcaaggaagt
     3721 ttcaaagagc tacccaatat ctgaggggcc tgagagagcc aacgaattag tagaatccta
     3781 tagaaaggct tcaaataaag c
//
