LOCUS       sequence-384567-        6393 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses I-WcaI homing endonuclease from Wickerhamomyces canadensis
            containing the K125I 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) K125I
FEATURES             Location/Qualifiers
     source          1..6393
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     534..553
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(573..650)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     856..875
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     959..978
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        959..977
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    978..1002
                     /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             1017..1039
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1058..1075
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1085..1102
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     primer_bind     complement(1852..1870)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1866..1913
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2088..2093,2094..2110,2111..2116))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2143..2161)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2229..2333
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2334..2402,2403..3194)
                     /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(2552..2571)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3365..3953
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3854..3873
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4107..4124
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4139..4278
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4204..4223)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4364..4386
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4380..4571)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5883..6393,1..572))
                     /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 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
       61 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
      121 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
      181 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
      241 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
      301 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
      361 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
      421 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
      481 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
      541 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
      601 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
      661 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
      721 gagcaccgcc gccgcaagga atggtgcatg caaggagatg gcgcccaaca gtcccccggc
      781 cacggggcct gccaccatac ccacgccgaa acaagcgctc atgagcccga agtggcgagc
      841 ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc
      901 ggtgatgccg gccacgatgc gtccggcgta gaggatcgag atctcgatcc cgcgaaatta
      961 atacgactca ctatagggga attgtgagcg gataacaatt cccctctaga aataattttg
     1021 tttaacttta agaaggagat ataccatggg cagcagccat catcatcatc atcacagcag
     1081 cggcctggtg ccgcgcggca gccatatgat gaaaaaacag attatcaaca aaaaagacct
     1141 gctgggcctg ggtccgaact caaaactgat taaagactac aaaaaacaat ggacgaccct
     1201 gagtaaaatt caggaagaaa cgctgattgg caacatcctg ggtgatgtct acatcaaaaa
     1261 actgaaacgt aacaaacatt ttctgctgca attcgaatgg aaaaacaaag catacatcga
     1321 acacatcgtg cgcgtttttg acgaatatgt tatttccccg ccgaccctgt acgaacgtaa
     1381 aaaccaccta ggcaataaag tcatcacctg gcgcgcgcag acgtttgaac ataaagcctt
     1441 cgataaactg ggttactact tcatggaaaa ccacaagatt attatcaaac cggacctggt
     1501 gctgaattat attacggaac gtagcctggc gtactggttt atggatgacg gcggtaaatg
     1561 ggattacaac aagaaaacca aaaacaaatc tctggtcctg cacacgcaag gcttcaaaaa
     1621 agaagaagtg gaaatcctga tcaacgatct gaacatcaaa ttcaacctga actgcagcat
     1681 caaattcaac aaaaacaaac cgatcatcta catcccgaac aaagactacg aactgtttta
     1741 caacctggtt aatccgtaca ttatcccgga aatgaaatac aaactgctgt tcaatgtgta
     1801 aggatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc accgctgagc
     1861 aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt ttgctgaaag
     1921 gaggaactat atccggatat cccgcaagag gcccggcagt accggcataa ccaagcctat
     1981 gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt tagatttcat
     2041 acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt aaagcttatc
     2101 gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg atacgcctat
     2161 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     2221 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2281 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     2341 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     2401 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     2461 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     2521 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg
     2581 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     2641 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     2701 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     2761 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     2821 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag
     2881 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     2941 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     3001 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     3061 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     3121 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     3181 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     3241 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     3301 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     3361 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     3421 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     3481 gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc
     3541 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     3601 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     3661 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     3721 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     3781 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     3841 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     3901 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     3961 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     4021 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     4081 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     4141 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct
     4201 cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt
     4261 gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
     4321 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
     4381 cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg
     4441 tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc
     4501 tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc
     4561 tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg
     4621 atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg
     4681 gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact
     4741 cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag
     4801 catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga
     4861 ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg
     4921 cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg
     4981 caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc
     5041 caggacccaa cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg
     5101 gatatgttct gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct
     5161 ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg
     5221 tggcccggct ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc
     5281 ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga
     5341 tcagcggtcc aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc
     5401 cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc
     5461 cgccggaagc gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca
     5521 gcaagacgta gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga
     5581 aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata
     5641 ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga
     5701 cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg
     5761 cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca
     5821 agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc
     5881 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     5941 aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt
     6001 gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg
     6061 tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata
     6121 taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc
     6181 agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc
     6241 atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg
     6301 gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta
     6361 tgccagccag ccagacgcag acgcgccgag aca
//
