LOCUS       sequence-256090-        5245 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes HA tagged human I-kappa-B-alpha(1-62) for purification by 
            GST affinity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5245)
  AUTHORS   Smolko CM, Janes KA
  TITLE     An ultrasensitive fiveplex activity assay for cellular kinases.
  JOURNAL   Sci Rep. 2019 Dec 19;9(1):19409. doi: 10.1038/s41598-019-55998-8.
  PUBMED    31857650
REFERENCE   2  (bases 1 to 5245)
  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: "Sci Rep."; 
            date: "2019-12-19"; volume: "9(1):19409. doi"; pages: " 
            10.1038/s41598-019-55998-8"
COMMENT     Sequence Label: pGEX-4T-1-3xHA-I-kappa-B-alpha(1-62)
FEATURES             Location/Qualifiers
     source          1..5245
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(45..50,51..66,67..73)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    81..97
                     /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)."
     CDS             120..773
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     731..753
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             780..797
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             801..881
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(1157..1179)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1324..1342)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1410..1514
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1515..1583,1584..2375)
                     /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(1733..1752)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2546..3134
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3035..3054
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3288..3305
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3378..3455
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3456..4538
                     /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
                     EDGTRLGVEHLVALGHQQIALLAGPSSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3475..3494)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4587..4592,4593..4610,4611..4617)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4625..4641
                     /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)."
     primer_bind     4649..4665
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4649..4665
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4661..5188
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4681..4698)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4681..4697)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4690..4712)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca attaatcatc
       61 ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagtattca
      121 tgtcccctat actaggttat tggaaaatta agggccttgt gcaacccact cgacttcttt
      181 tggaatatct tgaagaaaaa tatgaagagc atttgtatga gcgcgatgaa ggtgataaat
      241 ggcgaaacaa aaagtttgaa ttgggtttgg agtttcccaa tcttccttat tatattgatg
      301 gtgatgttaa attaacacag tctatggcca tcatacgtta tatagctgac aagcacaaca
      361 tgttgggtgg ttgtccaaaa gagcgtgcag agatttcaat gcttgaagga gcggttttgg
      421 atattagata cggtgtttcg agaattgcat atagtaaaga ctttgaaact ctcaaagttg
      481 attttcttag caagctacct gaaatgctga aaatgttcga agatcgttta tgtcataaaa
      541 catatttaaa tggtgatcat gtaacccatc ctgacttcat gttgtatgac gctcttgatg
      601 ttgttttata catggaccca atgtgcctgg atgcgttccc aaaattagtt tgttttaaaa
      661 aacgtattga agctatccca caaattgata agtacttgaa atccagcaag tatatagcat
      721 ggcctttgca gggctggcaa gccacgtttg gtggtggcga ccatcctcca aaatcggatc
      781 tggttccgcg tggatctatg tacccatacg atgttccaga ttacgcttac ccatacgatg
      841 ttccagatta cgcttaccca tacgatgttc cagattacgc tggatccatg ttccaggcgg
      901 ccgagcgccc ccaggagtgg gccatggagg gcccccgcga cgggctgaag aaggagcggc
      961 tactggacga ccgccacgac agcggcctgg actccatgaa agacgaggag tacgagcaga
     1021 tggtcaagga gctgcaggag atccgcctcg agccgcagga ggtgccgcgc ggctgagaat
     1081 tcccgggtcg actcgagcgg ccgcatcgtg actgactgac gatctgcctc gcgcgtttcg
     1141 gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt
     1201 aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc
     1261 ggggcgcagc catgacccag tcacgtagcg atagcggagt gtataattct tgaagacgaa
     1321 agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga
     1381 cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     1441 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     1501 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     1561 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     1621 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     1681 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     1741 gcgcggtatt atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt
     1801 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     1861 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     1921 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     1981 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     2041 gtgacaccac gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac
     2101 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     2161 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     2221 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     2281 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     2341 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     2401 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     2461 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     2521 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     2581 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     2641 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag
     2701 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     2761 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     2821 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     2881 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     2941 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     3001 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     3061 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     3121 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     3181 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     3241 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     3301 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3361 cacaccgcat aaattccgac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat
     3421 agcgcccgga agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg
     3481 tcgcagagta tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc
     3541 acgtttctgc gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc
     3601 ccaaccgcgt ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct
     3661 ccagtctggc cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc
     3721 aactgggtgc cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag
     3781 cggcggtgca caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg
     3841 atgaccagga tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg
     3901 atgtctctga ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac
     3961 tgggcgtgga gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat
     4021 caagttctgt ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc
     4081 aaattcagcc gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa
     4141 ccatgcaaat gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga
     4201 tggcgctggg cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct
     4261 cggtagtggg atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca
     4321 tcaaacagga ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc
     4381 agggccaggc ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca
     4441 ccctggcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     4501 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt
     4561 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     4621 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacggattc
     4681 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
     4741 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
     4801 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc tttgcctggt ttccggcacc
     4861 agaagcggtg ccggaaagct ggctggagtg cgatcttcct gaggccgata ctgtcgtcgt
     4921 cccctcaaac tggcagatgc acggttacga tgcgcccatc tacaccaacg tgacctatcc
     4981 cattacggtc aatccgccgt ttgttcccac ggagaatccg acgggttgtt actcgctcac
     5041 atttaatgtt gatgaaagct ggctacagga aggccagacg cgaattattt ttgatggcgt
     5101 tggaattagc ttatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga
     5161 agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca
     5221 ctcccgttct ggataatgtt ttttg
//
