LOCUS       sequence-383673-        5002 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PI-SceI (VDE) homing endonuclease/intein from the T7 
            promoter that contains the K301A active site mutation.
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 K301A
FEATURES             Location/Qualifiers
     source          1..5002
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1191..1202
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1336..1353
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1406..1424)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1420..1467
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1504..1959
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1591..1610)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1801..1822
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1985..2089
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2090..2158,2159..2950)
                     /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(2308..2327)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3121..3709
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3610..3629
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3863..3880
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3895..4034
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3960..3979)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4120..4142
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4136..4327)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     4836..4855
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4836..4854
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             4886..4908
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 ggtaaagatg gcagacctcg tgaggtaatt aaattgccca gaggaagaga aactatgtac
       61 agcgtcgtgc agaaaagtca gcacagagcc cacaaaagtg actcaagtcg tgaagtgcca
      121 gaattactca agtttacgtg taatgcgacc catgaactag ttgttagaac acctcgtagt
      181 gtccgccgtt tgtctcgtac cattaagggt gtcgaatatt ttgaagttat tacttttgag
      241 atgggccaaa agaaagcccc cgacggtaga attgttgagc ttgtcaagga agtttcaaag
      301 agctacccaa tatctgaggg gcccgagaga gccaacgaat tagtagaatc ctatagaaag
      361 gcttcaaata aagcttattt tgagtggact attgaggcgc gcgatctttc tctgttgggt
      421 tcccatgttc gtaaagctac ctaccagact tacgctccaa ttctttatga gaatgaccac
      481 tttttcgact acatgcaaaa aagtaagttt catctcacca ttgaaggtcc aaaagtactt
      541 gcttatctcc taggtttatg gattggtgat ggattgtcgg accgggcaac tttttcggtt
      601 gattccagag atacttcttt gatggaacgg gttaccgaat atgctgaaaa gttgaatttg
      661 tgcgccgagt ataaggacag aaaagaacca caagttgcca aaactgttaa tttgtactct
      721 aaagtcgtac gaggtaatgg tattcgcaat aatcttaata ctgagaatcc attatgggac
      781 gctattgttg gcttaggatt cttgaaggac ggtgtcgcta atattccttc tttcttgtct
      841 acggacaata tcggtacgcg tgaaacattt ctggccggtc taattgattc tgatggctac
      901 gtaactgatg agcatggtat taaagcaaca ataaagacaa ttcatacttc tgtcagagat
      961 ggtttggttt cccttgctcg ttctttaggc ttagtagtct cggttaacgc agaacctgct
     1021 aaggttgaca tgaatggcac caaacataaa attagttatg ctatttatat gtctggtgga
     1081 gatgttttgc ttaacgttct ttcgaagtgt gccggctcta aaaaattcag gcctgctccc
     1141 gccgctgctt ttgcacgtga gtgccgcgga ttttatttcg agttacaaga attgaaggaa
     1201 gacgattatt atgggattac tttatctgat gattctgatc atcagttttt gcttgccaac
     1261 caggttgtcg tccatgcaaa atgggtcgcg gatccgaatt cgagctccgt cgacaagctt
     1321 gcggccgcac tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga
     1381 aaggaagctg agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc
     1441 tctaaacggg tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat
     1501 gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     1561 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     1621 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     1681 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
     1741 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
     1801 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
     1861 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
     1921 ttaacgcgaa ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa
     1981 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     2041 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     2101 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     2161 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     2221 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     2281 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
     2341 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     2401 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     2461 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     2521 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     2581 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     2641 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     2701 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     2761 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     2821 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     2881 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     2941 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     3001 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     3061 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     3121 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     3181 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     3241 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     3301 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     3361 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     3421 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     3481 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     3541 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     3601 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     3661 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     3721 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     3781 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     3841 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     3901 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     3961 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     4021 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     4081 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     4141 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     4201 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     4261 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     4321 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     4381 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     4441 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     4501 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     4561 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     4621 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     4681 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     4741 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     4801 acccaacgct gcccgagatc tcgatcccgc gaaattaata cgactcacta tagggagacc
     4861 acaacggttt ccctctagaa ataattttgt ttaactttaa gaaggagata tacatatgtg
     4921 ctttgccaag ggtaccaatg ttttaatggc ggatgggtct attgaatgta ttgaaaacat
     4981 tgaggttggt aataaggtca tg
//
