LOCUS       sequence-384566-        5002 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses PI-SceI (VDE) homing endonuclease/intein from the T7 
            promoter that contains the K403A active site mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5002)
  AUTHORS   Gimble FS, Duan X, Hu D, Quiocho FA
  TITLE     Identification of Lys-403 in the PI-SceI homing endonuclease as part
            of a symmetric catalytic center.
  JOURNAL   J Biol Chem. 1998 Nov 13;273(46):30524-9. doi: 
            10.1074/jbc.273.46.30524.
  PUBMED    9804821
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.273.46"; journalName: "J Biol Chem"; date: "1998-11-13-
            13"; volume: "273"; issue: "46"; pages: "30524-9"
COMMENT     Sequence Label: pET PI-SceI C-His K403A
FEATURES             Location/Qualifiers
     source          1..5002
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     258..277
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        258..276
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             308..330
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1615..1626
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             1760..1777
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1830..1848)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1844..1891
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1928..2383
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2015..2034)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2225..2246
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2409..2513
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2514..2582,2583..3374)
                     /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(2732..2751)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3545..4133
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4034..4053
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4287..4304
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4319..4458
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4384..4403)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4544..4566
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4560..4751)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
ORIGIN
        1 acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga
       61 agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc
      121 gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg
      181 tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga
      241 tctcgatccc gcgaaattaa tacgactcac tatagggaga ccacaacggt ttccctctag
      301 aaataatttt gtttaacttt aagaaggaga tatacatatg tgctttgcca agggtaccaa
      361 tgttttaatg gcggatgggt ctattgaatg tattgaaaac attgaggttg gtaataaggt
      421 catgggtaaa gatggcagac ctcgtgaggt aattaaattg cccagaggaa gagaaactat
      481 gtacagcgtc gtgcagaaaa gtcagcacag agcccacaaa agtgactcaa gtcgtgaagt
      541 gccagaatta ctcaagttta cgtgtaatgc gacccatgaa ctagttgtta gaacacctcg
      601 tagtgtccgc cgtttgtctc gtaccattaa gggtgtcgaa tattttgaag ttattacttt
      661 tgagatgggc caaaagaaag cccccgacgg tagaattgtt gagcttgtca aggaagtttc
      721 aaagagctac ccaatatctg aggggcccga gagagccaac gaattagtag aatcctatag
      781 aaaggcttca aataaagctt attttgagtg gactattgag gcgcgcgatc tttctctgtt
      841 gggttcccat gttcgtaaag ctacctacca gacttacgct ccaattcttt atgagaatga
      901 ccactttttc gactacatgc aaaaaagtaa gtttcatctc accattgaag gtccaaaagt
      961 acttgcttat ctcctaggtt tatggattgg tgatggattg tcggaccggg caactttttc
     1021 ggttgattcc agagatactt ctttgatgga acgggttacc gaatatgctg aaaagttgaa
     1081 tttgtgcgcc gagtataagg acagaaaaga accacaagtt gccaaaactg ttaatttgta
     1141 ctctaaagtc gtacgaggta atggtattcg caataatctt aatactgaga atccattatg
     1201 ggacgctatt gttggcttag gattcttgaa ggacggtgtc aaaaatattc cttctttctt
     1261 gtctacggac aatatcggta cgcgtgaaac atttctggcc ggtctaattg attctgatgg
     1321 ctacgtaact gatgagcatg gtattaaagc aacaataaag acaattcata cttctgtcag
     1381 agatggtttg gtttcccttg ctcgttcttt aggcttagta gtctcggtta acgcagaacc
     1441 tgctaaggtt gacatgaatg gcaccaaaca taaaattagt tatgctattt atatgtctgg
     1501 tggagatgtt ttgcttaacg ttctttcgaa gtgtgccggc tctaaagcat tcaggcctgc
     1561 tcccgccgct gcttttgcac gtgagtgccg cggattttat ttcgagttac aagaattgaa
     1621 ggaagacgat tattatggga ttactttatc tgatgattct gatcatcagt ttttgcttgc
     1681 caaccaggtt gtcgtccatg caaaatgggt cgcggatccg aattcgagct ccgtcgacaa
     1741 gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg ctaacaaagc
     1801 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg
     1861 ggcctctaaa cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggattggc
     1921 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     1981 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     2041 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     2101 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     2161 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     2221 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     2281 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     2341 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     2401 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2461 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2521 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2581 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2641 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2701 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     2761 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     2821 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     2881 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     2941 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     3001 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     3061 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     3121 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3181 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3241 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3301 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3361 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     3421 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3481 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3541 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3601 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3661 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3721 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3781 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3841 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3901 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3961 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     4021 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     4081 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     4141 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4201 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4261 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4321 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     4381 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     4441 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4501 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4561 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4621 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4681 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4741 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4801 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4861 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4921 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4981 catcctgcga tgcagatccg ga
//
