LOCUS       sequence-217724-        5769 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 5769)
  TITLE     Cwc25 plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5769)
  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            "Unpublished"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: pET29b-Cwc25
FEATURES             Location/Qualifiers
     source          1..5769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     30..49
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     137..156
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        137..155
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    156..180
                     /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             195..217
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             766..783
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(836..854)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      850..897
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      934..1389
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1021..1040)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1231..1252
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1481..2296)
                     /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(1481..2296)
                     /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     2204..2223
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2288..2307
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2418..3006
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2907..2926
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3160..3177
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3192..3331
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3257..3276)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3417..3439
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3433..3624)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4433..5515)
                     /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"
     primer_bind     5477..5496
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5516..5593)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc
       61 gcacctgtgg cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat
      121 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc
      181 cctctagaaa taattttgtt taactttaag aaggagatat acatggggtc gggcgatttg
      241 aatctgttga aatcatggaa tcccaagctt atgaaaaata gaaaaaaagt atgggagacc
      301 gaacaggatc taattaccga acagcagaag cttaacacgc gattgaaaga aattgaaaaa
      361 gaacgagaac taaatgaact gctgaacgaa tcgagcaagg ataagccgga gactctcaag
      421 aatgacttgg ctttgaagaa gtcgggcttg gaatggatgt atcaggatgc aaaactgagt
      481 gacgaaaagg aagactattt attaggtaag aaaaaactag attcatctat actaaaccaa
      541 cctgcaacac cgcctgtaag ggcggccact actatttccg cttctggtgc cgcaacatcc
      601 ataagttcgc agaagaagaa gtcaaaattg cttaaagatg atccaatgag taagtttaag
      661 gtaactaaac aacaaagaag aactccagac tcaacaaaaa aaagagcaat gtcgcaacga
      721 gggaaaccgt tgtccaagcc cgctccggac ctagactacc tcgagcacca ccaccaccac
      781 cactgagatc cggctgctaa caaagcccga aaggaagctg agttggctgc tgccaccgct
      841 gagcaataac tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg
      901 aaaggaggaa ctatatccgg attggcgaat gggacgcgcc ctgtagcggc gcattaagcg
      961 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg
     1021 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     1081 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     1141 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     1201 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     1261 tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt
     1321 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgc
     1381 ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt
     1441 ctaaatacat tcaaatatgt atccgctcat gaattaattc ttagaaaaac tcatcgagca
     1501 tcaaatgaaa ctgcaattta ttcatatcag gattatcaat accatatttt tgaaaaagcc
     1561 gtttctgtaa tgaaggagaa aactcaccga ggcagttcca taggatggca agatcctggt
     1621 atcggtctgc gattccgact cgtccaacat caatacaacc tattaatttc ccctcgtcaa
     1681 aaataaggtt atcaagtgag aaatcaccat gagtgacgac tgaatccggt gagaatggca
     1741 aaagtttatg catttctttc cagacttgtt caacaggcca gccattacgc tcgtcatcaa
     1801 aatcactcgc atcaaccaaa ccgttattca ttcgtgattg cgcctgagcg agacgaaata
     1861 cgcgatcgct gttaaaagga caattacaaa caggaatcga atgcaaccgg cgcaggaaca
     1921 ctgccagcgc atcaacaata ttttcacctg aatcaggata ttcttctaat acctggaatg
     1981 ctgttttccc ggggatcgca gtggtgagta accatgcatc atcaggagta cggataaaat
     2041 gcttgatggt cggaagaggc ataaattccg tcagccagtt tagtctgacc atctcatctg
     2101 taacatcatt ggcaacgcta cctttgccat gtttcagaaa caactctggc gcatcgggct
     2161 tcccatacaa tcgatagatt gtcgcacctg attgcccgac attatcgcga gcccatttat
     2221 acccatataa atcagcatcc atgttggaat ttaatcgcgg cctagagcaa gacgtttccc
     2281 gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac agttttattg
     2341 ttcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
     2401 aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca
     2461 aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt
     2521 ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg
     2581 tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc
     2641 ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
     2701 cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
     2761 agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc
     2821 gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca
     2881 ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg
     2941 tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
     3001 tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct
     3061 cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag
     3121 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
     3181 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3241 aatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc
     3301 gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc
     3361 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg
     3421 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agctgcggta
     3481 aagctcatca gcgtggtcgt gaagcgattc acagatgtct gcctgttcat ccgcgtccag
     3541 ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg ataaagcggg ccatgttaag
     3601 ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa gggggatttc tgttcatggg
     3661 ggtaatgata ccgatgaaac gagagaggat gctcacgata cgggttactg atgatgaaca
     3721 tgcccggtta ctggaacgtt gtgagggtaa acaactggcg gtatggatgc ggcgggacca
     3781 gagaaaaatc actcagggtc aatgccagcg cttcgttaat acagatgtag gtgttccaca
     3841 gggtagccag cagcatcctg cgatgcagat ccggaacata atggtgcagg gcgctgactt
     3901 ccgcgtttcc agactttacg aaacacggaa accgaagacc attcatgttg ttgctcaggt
     3961 cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg cgtatcggtg attcattctg
     4021 ctaaccagta aggcaacccc gccagcctag ccgggtcctc aacgacagga gcacgatcat
     4081 gcgcacccgt ggggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     4141 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     4201 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     4261 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     4321 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     4381 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     4441 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4501 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     4561 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     4621 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     4681 tgtcttcggt atcgtcgtat cccactaccg agatgtccgc accaacgcgc agcccggact
     4741 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     4801 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     4861 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     4921 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     4981 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     5041 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     5101 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     5161 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     5221 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     5281 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     5341 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     5401 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     5461 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     5521 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     5581 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     5641 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     5701 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     5761 ctcatgagc
//
