LOCUS       sequence-327673-        6510 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human KANK2 Ank domain - A670V mutant in bacterial cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6510)
  TITLE     Bacterial expression plasmids for production of recombinant KANK 
            proteins
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6510)
  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            "Unpublished"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: human KANK2-A670V Ankyrin Repeats pET151
FEATURES             Location/Qualifiers
     source          1..6510
                     /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(1211..1229)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1225..1272
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1510..1528)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1596..1700
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1701..1769,1770..2561)
                     /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(1919..1938)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2732..3320
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3221..3240
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3474..3491
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3506..3645
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3571..3590)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3731..3753
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3747..3938)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5250..6332)
                     /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     6294..6313
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6333..6410)
                     /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 attgatccct tcaccgaaga agaaattcgc atggaactga
      421 gtccggatct gattagcgca tgtctggcac tggaaaaata tctggataat ccgaatgcac
      481 tgaccgaacg tgaactgaaa gttgcatata ccaccgttct gcaagaatgg ctgcgtctgg
      541 catgtcgtag tgatgcacat ccggaactgg ttcgtcgtca tctggttacc tttcgtgcaa
      601 tgagcgcacg tctgctggat tatgttgtta atattgcaga tagcaatggc aacaccgtac
      661 tgcattatag cgttagccat gcaaattttc cggttgttca gcaactgctg gatagcggtg
      721 tttgtaaagt tgataaacag aatcgtgcag gttatagccc gattatgctg accgcactgg
      781 caaccctgaa aacccaggat gatattgaaa ccgtgctgca gctgtttcgt ctgggtaaca
      841 ttaatgcaaa agcaagccag gcaggtcaga cagcactgat gctggcagtg agccatggtc
      901 gtgttgatgt tgtgaaagca ctgctggcat gtgaagcaga tgttaatgtt caggatgatg
      961 atggtagtac cgcactgatg tgtgcatgcg aacatggtca taaagaaatt gcaggtctgc
     1021 tgctggccgt tccgagctgt gatattagcc tgacagatcg tgatggttca acagccctga
     1081 tggttgcact ggatgccggt cagagcgaaa ttgcaagcat gctgtatagc cgtatgaaca
     1141 tcaaataaaa gggcgagctc agatccggct gctaacaaag cccgaaagga agctgagttg
     1201 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     1261 aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg cccggcagta
     1321 ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg atgacgatga
     1381 gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac
     1441 taccgcatta aagctagctt atcgatgata agctgtcaaa catgagaatt aattcttgaa
     1501 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     1561 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1621 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1681 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1741 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1801 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1861 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1921 tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt cgccgcatac
     1981 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2041 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2101 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2161 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2221 acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
     2281 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2341 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2401 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2461 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2521 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2581 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2641 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2701 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2761 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2821 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc
     2881 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2941 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     3001 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     3061 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     3121 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     3181 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     3241 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     3301 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     3361 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     3421 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     3481 cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg
     3541 gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag
     3601 ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca
     3661 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
     3721 gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg
     3781 aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt
     3841 tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag
     3901 cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga
     3961 tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt
     4021 actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg
     4081 atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat
     4141 gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg
     4201 cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat
     4261 gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc
     4321 ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac
     4381 aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat gcgccgcgtg
     4441 cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt ttgcgcattc
     4501 acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc gttagcgagg
     4561 tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga cgcaacgcgg
     4621 ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc catgtgctcg
     4681 ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa
     4741 gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
     4801 tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata atggggaagg
     4861 ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg gccgccatgc
     4921 cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg acgaaggctt
     4981 gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc gtcgcgctcc
     5041 agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt cctacgagtt
     5101 gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc gcccaccgga
     5161 aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt gcctaatgag
     5221 tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5281 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5341 gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc cttcaccgcc
     5401 tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag gcgaaaatcc
     5461 tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc gtcgtatccc
     5521 actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg cattgcgccc
     5581 agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc attcagcatt
     5641 tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc cgctatcggc
     5701 tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg cgccgagaca
     5761 gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc
     5821 acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg tgtctggtca
     5881 gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc aatggcatcc
     5941 tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag aagattgtgc
     6001 accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac cacgctggca
     6061 cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc
     6121 agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg ttgtgccacg
     6181 cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg cgttttcgca
     6241 gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac accggcatac
     6301 tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg actctcttcc
     6361 gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg
     6421 acgctctccc ttatgcgact cctgcattag gaagcagccc agtagtaggt tgaggccgtt
     6481 gagcaccgcc gccgcaagga atggtgcatg
//
