LOCUS       sequence-383258-        5002 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PI-SceI (VDE) homing endonuclease/intein from the T7 
            promoter with a carboxyl-terminal histidine tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5002)
  AUTHORS   He Z, Crist M, Yen H, Duan X, Quiocho FA, Gimble FS
  TITLE     Amino acid residues in both the protein splicing and endonuclease 
            domains of the PI-SceI intein mediate DNA binding.
  JOURNAL   J Biol Chem. 1998 Feb 20;273(8):4607-15. doi: 
            10.1074/jbc.273.8.4607.
  PUBMED    9468518
REFERENCE   2  (bases 1 to 5002)
  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.273.8";
            journalName: "J Biol Chem"; date: "1998-02-20- 20"; volume: "273"; 
            issue: "8"; pages: "4607-15"
COMMENT     Sequence Label: pET PI-SceI C-His
FEATURES             Location/Qualifiers
     source          1..5002
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     402..421
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        402..420
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             452..474
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1759..1770
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1904..1921
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1974..1992)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1988..2035
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2072..2527
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2159..2178)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2369..2390
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2553..2657
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2658..2726,2727..3518)
                     /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(2876..2895)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3689..4277
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4178..4197
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4431..4448
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4463..4602
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4528..4547)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4688..4710
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4704..4895)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
       61 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
      121 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
      181 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
      241 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
      301 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg
      361 gccaggaccc aacgctgccc gagatctcga tcccgcgaaa ttaatacgac tcactatagg
      421 gagaccacaa cggtttccct ctagaaataa ttttgtttaa ctttaagaag gagatataca
      481 tatgtgcttt gccaagggta ccaatgtttt aatggcggat gggtctattg aatgtattga
      541 aaacattgag gttggtaata aggtcatggg taaagatggc agacctcgtg aggtaattaa
      601 attgcccaga ggaagagaaa ctatgtacag cgtcgtgcag aaaagtcagc acagagccca
      661 caaaagtgac tcaagtcgtg aagtgccaga attactcaag tttacgtgta atgcgaccca
      721 tgaactagtt gttagaacac ctcgtagtgt ccgccgtttg tctcgtacca ttaagggtgt
      781 cgaatatttt gaagttatta cttttgagat gggccaaaag aaagcccccg acggtagaat
      841 tgttgagctt gtcaaggaag tttcaaagag ctacccaata tctgaggggc ccgagagagc
      901 caacgaatta gtagaatcct atagaaaggc ttcaaataaa gcttattttg agtggactat
      961 tgaggcgcgc gatctttctc tgttgggttc ccatgttcgt aaagctacct accagactta
     1021 cgctccaatt ctttatgaga atgaccactt tttcgactac atgcaaaaaa gtaagtttca
     1081 tctcaccatt gaaggtccaa aagtacttgc ttatctccta ggtttatgga ttggtgatgg
     1141 attgtcggac cgggcaactt tttcggttga ttccagagat acttctttga tggaacgggt
     1201 taccgaatat gctgaaaagt tgaatttgtg cgccgagtat aaggacagaa aagaaccaca
     1261 agttgccaaa actgttaatt tgtactctaa agtcgtacga ggtaatggta ttcgcaataa
     1321 tcttaatact gagaatccat tatgggacgc tattgttggc ttaggattct tgaaggacgg
     1381 tgtcaaaaat attccttctt tcttgtctac ggacaatatc ggtacgcgtg aaacatttct
     1441 ggccggtcta attgattctg atggctacgt aactgatgag catggtatta aagcaacaat
     1501 aaagacaatt catacttctg tcagagatgg tttggtttcc cttgctcgtt ctttaggctt
     1561 agtagtctcg gttaacgcag aacctgctaa ggttgacatg aatggcacca aacataaaat
     1621 tagttatgct atttatatgt ctggtggaga tgttttgctt aacgttcttt cgaagtgtgc
     1681 cggctctaaa aaattcaggc ctgctcccgc cgctgctttt gcacgtgagt gccgcggatt
     1741 ttatttcgag ttacaagaat tgaaggaaga cgattattat gggattactt tatctgatga
     1801 ttctgatcat cagtttttgc ttgccaacca ggttgtcgtc catgcaaaat gggtcgcgga
     1861 tccgaattcg agctccgtcg acaagcttgc ggccgcactc gagcaccacc accaccacca
     1921 ctgagatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga
     1981 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     2041 aggaggaact atatccggat tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     2101 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     2161 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     2221 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     2281 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     2341 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     2401 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     2461 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     2521 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     2581 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     2641 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     2701 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     2761 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     2821 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     2881 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     2941 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     3001 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     3061 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     3121 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     3181 agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     3241 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     3301 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     3361 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     3421 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     3481 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     3541 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     3601 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     3661 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     3721 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     3781 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
     3841 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     3901 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     3961 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     4021 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     4081 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     4141 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     4201 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     4261 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     4321 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     4381 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     4441 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4501 tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4561 gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca cccgccaaca
     4621 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     4681 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg
     4741 cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca
     4801 tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct gataaagcgg
     4861 gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta agggggattt
     4921 ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat acgggttact
     4981 gatgatgaac atgcccggtt ac
//
