LOCUS       sequence-217722-        5838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  his tagged Cwc25.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5838)
  TITLE     Cwc25 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5838)
  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"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pET28B-Cwc25
FEATURES             Location/Qualifiers
     source          1..5838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     131..150
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     234..253
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        234..252
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    253..277
                     /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             292..314
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             333..350
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             360..377
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             936..953
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1006..1024)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1020..1067
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1104..1559
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1191..1210)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1401..1422
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1651..2466)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1651..2466)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2374..2393
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2458..2477
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2588..3176
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3077..3096
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3330..3347
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3362..3501
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3427..3446)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3587..3609
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3603..3794)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4603..5685)
                     /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="MKPVTLYDVAEYAGVSYQTVSRVVNQSSHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5647..5666
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5686..5763)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc ccggccacgg
       61 ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat
      121 cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg gcgccggtga
      181 tgccggccac gatgcgtccg gcgtagagga tcgagatctc gatcccgcga aattaatacg
      241 actcactata ggggaattgt gagcggataa caattcccct ctagaaataa ttttgtttaa
      301 ctttaagaag gagatatacc atgggcagca gccatcatca tcatcatcac agcagcggcc
      361 tggtgccgcg cggcagccat atggggtcgg gcgatttgaa tctgttgaaa tcatggaatc
      421 ccaagcttat gaaaaataga aaaaaagtat gggagaccga acaggatcta attaccgaac
      481 agcagaagct taacacgcga ttgaaagaaa ttgaaaaaga acgagaacta aatgaactgc
      541 tgaacgaatc gagcaaggat aagccggaga ctctcaagaa tgacttggct ttgaagaagt
      601 cgggcttgga atggatgtat caggatgcaa aactgagtga cgaaaaggaa gactatttat
      661 taggtaagaa aaaactagat tcatctatac taaaccaacc tgcaacaccg cctgtaaggg
      721 cggccactac tatttccgct tctggtgccg caacatccat aagttcgcag aagaagaagt
      781 caaaattgct taaagatgat ccaatgagta agtttaaggt aactaaacaa caaagaagaa
      841 ctccagactc aacaaaaaaa agagcaatgt cgcaacgagg gaaaccgttg tccaagcccg
      901 ctccggacct agactactga gcggccgcac tcgagcacca ccaccaccac cactgagatc
      961 cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     1021 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1081 ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     1141 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     1201 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     1261 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     1321 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     1381 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     1441 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     1501 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc ttacaattta
     1561 ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat
     1621 tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca tcaaatgaaa
     1681 ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc gtttctgtaa
     1741 tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt atcggtctgc
     1801 gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa aaataaggtt
     1861 atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca aaagtttatg
     1921 catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa aatcactcgc
     1981 atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata cgcgatcgct
     2041 gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca ctgccagcgc
     2101 atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg ctgttttccc
     2161 ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat gcttgatggt
     2221 cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg taacatcatt
     2281 ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct tcccatacaa
     2341 tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat acccatataa
     2401 atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc gttgaatatg
     2461 gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg ttcatgacca
     2521 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2581 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2641 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2701 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     2761 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2821 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     2881 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2941 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     3001 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3061 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3121 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3181 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3241 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3301 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     3361 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc aatggtgcac
     3421 tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc gctatcgcta
     3481 cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     3541 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg
     3601 tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta aagctcatca
     3661 gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag ctcgttgagt
     3721 ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag ggcggttttt
     3781 tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg ggtaatgata
     3841 ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca tgcccggtta
     3901 ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca gagaaaaatc
     3961 actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca gggtagccag
     4021 cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt ccgcgtttcc
     4081 agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt cgcagacgtt
     4141 ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg ctaaccagta
     4201 aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat gcgcacccgt
     4261 ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca
     4321 gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc
     4381 atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc
     4441 tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc
     4501 cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc
     4561 ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     4621 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     4681 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     4741 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     4801 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     4861 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     4921 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     4981 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     5041 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     5101 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     5161 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     5221 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     5281 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     5341 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     5401 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     5461 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     5521 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     5581 ccgcgttttc gcagaaacgt ggctggactg gttcaccacg cgggaaacgg tctgataaga
     5641 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     5701 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt
     5761 gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta
     5821 ggttgaggcc gttgagca
//
