LOCUS       sequence-384568-        6393 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses I-WcaI homing endonuclease from Wickerhamomyces canadensis
            containing the K226I mutation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6393)
  TITLE     Gimble lab plasmids
REFERENCE   2  (bases 1 to 6393)
  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"
COMMENT     Sequence Label: pET15b (I-WcaINhis) K226I
FEATURES             Location/Qualifiers
     source          1..6393
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     549..568
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(588..665)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     871..890
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     974..993
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        974..992
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    993..1017
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             1032..1054
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1073..1090
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1100..1117
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1867..1885)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1881..1928
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2103..2108,2109..2125,2126..2131))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2158..2176)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2244..2348
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2349..2417,2418..3209)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2567..2586)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3380..3968
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3869..3888
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4122..4139
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4154..4293
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4219..4238)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4379..4401
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4395..4586)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5898..6393,1..587))
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
ORIGIN
        1 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
       61 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
      121 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
      181 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
      241 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
      301 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
      361 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
      421 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
      481 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
      541 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
      601 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
      661 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
      721 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
      781 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
      841 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
      901 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatctc
      961 gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct
     1021 ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgggcagca gccatcatca
     1081 tcatcatcac agcagcggcc tggtgccgcg cggcagccat atgatgaaaa aacagattat
     1141 caacaaaaaa gacctgctgg gcctgggtcc gaactcaaaa ctgattaaag actacaaaaa
     1201 acaatggacg accctgagta aaattcagga agaaacgctg attggcaaca tcctgggtga
     1261 tgtctacatc aaaaaactga aacgtaacaa acattttctg ctgcaattcg aatggaaaaa
     1321 caaagcatac atcgaacaca tcgtgcgcgt ttttgacgaa tatgttattt ccccgccgac
     1381 cctgtacgaa cgtaaaaacc acctaggcaa taaagtcatc acctggcgcg cgcagacgtt
     1441 tgaacataaa gccttcgata aactgggtta ctacttcatg gaaaaccaca agaaaattat
     1501 caaaccggac ctggtgctga attatattac ggaacgtagc ctggcgtact ggtttatgga
     1561 tgacggcggt aaatgggatt acaacaagaa aaccaaaaac aaatctctgg tcctgcacac
     1621 gcaaggcttc aaaaaagaag aagtggaaat cctgatcaac gatctgaaca tcaaattcaa
     1681 cctgaactgc agcatcaaat tcaacaaaaa caaaccgatc atctacatcc cgaacaaaga
     1741 ctacgaactg ttttacaacc tggttaatcc gtacattatc ccggaaatga aatacattct
     1801 gctgttcaat gtgtaaggat ccggctgcta acaaagcccg aaaggaagct gagttggctg
     1861 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
     1921 gttttttgct gaaaggagga actatatccg gatatcccgc aagaggcccg gcagtaccgg
     1981 cataaccaag cctatgccta cagcatccag ggtgacggtg ccgaggatga cgatgagcgc
     2041 attgttagat ttcatacacg gtgcctgact gcgttagcaa tttaactgtg ataaactacc
     2101 gcattaaagc ttatcgatga taagctgtca aacatgagaa ttcttgaaga cgaaagggcc
     2161 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     2221 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     2281 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     2341 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     2401 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     2461 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     2521 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     2581 tattatcccg tgttgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     2641 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     2701 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     2761 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     2821 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     2881 ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     2941 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     3001 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     3061 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     3121 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     3181 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     3241 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     3301 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     3361 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     3421 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     3481 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     3541 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     3601 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     3661 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     3721 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     3781 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     3841 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     3901 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     3961 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     4021 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     4081 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     4141 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     4201 gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4261 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4321 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4381 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     4441 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     4501 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     4561 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     4621 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     4681 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     4741 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     4801 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     4861 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     4921 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     4981 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     5041 atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag
     5101 atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc
     5161 aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt
     5221 ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa
     5281 ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat
     5341 aaatcgccgt gacgatcagc ggtccaatga tcgaagttag gctggtaaga gccgcgagcg
     5401 atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg
     5461 cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc
     5521 gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg
     5581 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     5641 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     5701 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     5761 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     5821 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     5881 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5941 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     6001 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     6061 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     6121 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     6181 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     6241 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     6301 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     6361 cgagtgagat atttatgcca gccagccaga cgc
//
