LOCUS       sequence-327667-        6537 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses mouse KANK4 Ank domain -Y801H mutant in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6537)
  TITLE     Bacterial expression plasmids for production of recombinant KANK 
            proteins
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6537)
  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            "Unpublished"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: mouse KANK4-Y801H Ankyrin Repeats pET151
FEATURES             Location/Qualifiers
     source          1..6537
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     106..125
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     209..228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        209..227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    228..252
                     /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             267..289
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             300..317
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             318..359
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             360..380
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1238..1256)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1252..1299
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1537..1555)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1623..1727
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1728..1796,1797..2588)
                     /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(1946..1965)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2759..3347
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3248..3267
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3501..3518
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3533..3672
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3598..3617)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3758..3780
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3774..3965)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5277..6359)
                     /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     6321..6340
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6360..6437)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa
       61 acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat
      121 ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta
      181 gaggatcgag atctcgatcc cgcgaaatta atacgactca ctatagggga attgtgagcg
      241 gataacaatt cccctctaga aataattttg tttaacttta agaaggagat atacatatgc
      301 atcatcacca tcaccatggt aagcctatcc ctaaccctct cctcggtctc gattctacgg
      361 aaaacctgta ttttcaggga ggatccctga gggctgagag atataaaccc tcagaggaat
      421 ttcttaacgc gtgccagaca ctgagccagc acctgccgga aactggggat accaccaagc
      481 agctcctgag gcaaagcttg aataccatca gtcaggagtg gttccgtgtg tccagcagga
      541 agttatctag ccctgaggct gtggctgccc acctccttga ggtccagcct cactctccat
      601 atcttctgaa gctacttgtc aacctggctg accgaagtgg gaacacagcc cttcactaca
      661 gtgtgtcaca ctccaacttc gccattgtca agctactgct ggacacaggt gtctgcaatg
      721 tggatcatca gaacaaagct ggctacactg ctgtgatgat cactcccttg gcttctgctg
      781 agacaaaaga agacatggct gttgtctgga aactcttaag agaggggaat gtgaacatac
      841 aagcaactca gggaggacag actgcactga tgttaggagt cagccacgac cgggaggaca
      901 tggtccaagc actactgagc tgccaggcag atgtcaacct gcaggacaat gatggatctt
      961 ccgccctcat gctggcctgt caccaaggca acgccgacct ggtacgactg ctcctggcac
     1021 acccagcctg caacagcagc ctgactgaca aggctggccg aacagctttg tccctcgttc
     1081 tgaattcacc tgcccacgtg gaaatcgcag agcttctgcg ggcccactca gagccgggca
     1141 ggtccctggg gccgaaggag ctgcagaaga atctcgagta agagctcaga tccggctgct
     1201 aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     1261 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     1321 ggatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca
     1381 gggtgacggt gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac
     1441 tgcgttagca atttaactgt gataaactac cgcattaaag ctagcttatc gatgataagc
     1501 tgtcaaacat gagaattaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     1561 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     1621 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1681 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     1741 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     1801 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     1861 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1921 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     1981 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     2041 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     2101 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     2161 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     2221 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     2281 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     2341 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2401 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     2461 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     2521 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     2581 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     2641 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     2701 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2761 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2821 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2881 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2941 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     3001 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     3061 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     3121 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3181 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3241 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3301 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3361 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3421 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3481 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3541 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     3601 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3661 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3721 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3781 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     3841 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     3901 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     3961 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     4021 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     4081 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     4141 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     4201 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     4261 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     4321 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4381 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac
     4441 ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg
     4501 ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt
     4561 cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc
     4621 ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa
     4681 tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg
     4741 gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat
     4801 ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg
     4861 aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga
     4921 cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4981 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     5041 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     5101 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     5161 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     5221 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     5281 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5341 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     5401 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     5461 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     5521 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     5581 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     5641 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     5701 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5761 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5821 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5881 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5941 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     6001 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     6061 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     6121 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     6181 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     6241 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     6301 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     6361 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     6421 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     6481 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatg
//
