LOCUS       sequence-383257-        5027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PI-SceI (VDE) homing endonuclease/intein from the T7 
            promoter.
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
FEATURES             Location/Qualifiers
     source          1..5027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1250..1261
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1341..1370
                     /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             1420..1437
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1490..1508)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1504..1551
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1588..2043
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1675..1694)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1885..1906
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2069..2173
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2174..2242,2243..3034)
                     /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(2392..2411)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3205..3793
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3694..3713
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3947..3964
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3979..4118
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4044..4063)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4204..4226
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4220..4411)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4920..4939
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4920..4938
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4970..4992
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             join(5003..5027,1..794)
                     /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 taatggcgga tgggtctatt gaatgtattg aaaacattga ggttggtaat aaggtcatgg
       61 gtaaagatgg cagacctcgt gaggtaatta aattgcccag aggaagagaa actatgtaca
      121 gcgtcgtgca gaaaagtcag cacagagccc acaaaagtga ctcaagtcgt gaagtgccag
      181 aattactcaa gtttacgtgt aatgcgaccc atgagttggt tgttagaaca cctcgtagtg
      241 tccgccgttt gtctcgtacc attaagggtg tcgaatattt tgaagttatt acttttgaga
      301 tgggccaaaa gaaagccccc gacggtagaa ttgttgagct tgtcaaggaa gtttcaaaga
      361 gctacccaat atctgagggg cctgagagag ccaacgaatt agtagaatcc tatagaaagg
      421 cttcaaataa agcttatttt gagtggacta ttgaggccag agatctttct ctgttgggtt
      481 cccatgttcg taaagctacc taccagactt acgctccaat tctttatgag aatgaccact
      541 ttttcgacta catgcaaaaa agtaagtttc atctcaccat tgaaggtcca aaagtacttg
      601 cttatctcct aggtttatgg attggtgatg gattgtcgga ccgggcaact ttttcggttg
      661 attccagaga tacttctttg atggaacgtg ttactgaata tgctgaaaag ttgaatttgt
      721 gcgccgagta taaggacaga aaagaaccac aagttgccaa aactgttaat ttgtactcta
      781 aagttgtcag aggtaatggt attcgcaata atcttaatac tgagaatcca ttatgggacg
      841 ctattgttgg cttaggattc ttgaaggacg gtgtcaaaaa tattccttct ttcttgtcta
      901 cggacaatat cggtactcgt gaaacatttc tggccggtct aattgattct gatggctacg
      961 taactgatga gcatggtatt aaagcaacaa taaagacaat tcatacttct gtcagagatg
     1021 gtttggtttc ccttgctcgt tctttaggct tagtagtctc ggttaacgca gaacctgcta
     1081 aggttgacat gaatggcacc aaacataaaa ttagttatgc tatttatatg tctggtggag
     1141 atgttttgct taacgttctt tcgaagtgtg ccggctctaa aaaattcagg cctgctcccg
     1201 ccgctgcttt tgcacgtgag tgccgcggat tttatttcga gttacaagaa ttgaaggaag
     1261 acgattatta tgggattact ttatctgatg attctgatca tcagtttttg cttgccaacc
     1321 aggttgtcgt ccataattag gctagcatga ctggtggaca gcaaatgggt cgcggatccg
     1381 aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga
     1441 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1501 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1561 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     1621 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     1681 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     1741 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     1801 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     1861 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1921 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1981 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     2041 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2101 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2161 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2221 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2281 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2341 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     2401 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     2461 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2521 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     2581 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     2641 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     2701 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     2761 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     2821 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     2881 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     2941 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3001 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3061 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3121 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3181 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3241 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3301 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3361 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3421 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3481 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3541 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3601 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3661 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3721 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3781 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3841 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     3901 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     3961 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4021 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     4081 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4141 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4201 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4261 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     4321 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     4381 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     4441 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     4501 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     4561 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     4621 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     4681 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     4741 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     4801 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     4861 cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatctcgatc ccgcgaaatt
     4921 aatacgactc actataggga gaccacaacg gtttccctct agaaataatt ttgtttaact
     4981 ttaagaagga gatatacata tgtgctttgc caagggtacc aatgttt
//
