LOCUS       sequence-256106-        5062 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Empty backbone for HA tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5062)
  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 5062)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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
FEATURES             Location/Qualifiers
     source          1..5062
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     40..57
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        130..207
                     /gene="lacI"
                     /label=lacI promoter
     CDS             208..1290
                     /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     complement(227..246)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1339..1344,1345..1362,1363..1369)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1377..1393
                     /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     1401..1417
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1401..1417
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1413..1940
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(1433..1450)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1433..1449)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1442..1464)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(2042..2047,2048..2063,2064..2070)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2078..2094
                     /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             2117..2770
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2728..2750
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2777..2794
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2798..2878
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(2971..2993)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3138..3156)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3224..3328
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3329..3397,3398..4189)
                     /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(3547..3566)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4360..4948
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4849..4868
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa
       61 gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
      121 ataaattccg acaccatcga atggcgcaaa acctttcgcg gtatggcatg atagcgcccg
      181 gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa cgttatacga tgtcgcagag
      241 tatgccggtg tctcttatca gaccgtttcc cgcgtggtga accaggccag ccacgtttct
      301 gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc tgaattacat tcccaaccgc
      361 gtggcacaac aactggcggg caaacagtcg ttgctgattg gcgttgccac ctccagtctg
      421 gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat ctcgcgccga tcaactgggt
      481 gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg aagcctgtaa agcggcggtg
      541 cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta actatccgct ggatgaccag
      601 gatgccattg ctgtggaagc tgcctgcact aatgttccgg cgttatttct tgatgtctct
      661 gaccagacac ccatcaacag tattattttc tcccatgaag acggtacgcg actgggcgtg
      721 gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt tagcgggccc attaagttct
      781 gtctcggcgc gtctgcgtct ggctggctgg cataaatatc tcactcgcaa tcaaattcag
      841 ccgatagcgg aacgggaagg cgactggagt gccatgtccg gttttcaaca aaccatgcaa
      901 atgctgaatg agggcatcgt tcccactgcg atgctggttg ccaacgatca gatggcgctg
      961 ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg gtgcggatat ctcggtagtg
     1021 ggatacgacg ataccgaaga cagctcatgt tatatcccgc cgttaaccac catcaaacag
     1081 gattttcgcc tgctggggca aaccagcgtg gaccgcttgc tgcaactctc tcagggccag
     1141 gcggtgaagg gcaatcagct gttgcccgtc tcactggtga aaagaaaaac caccctggcg
     1201 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
     1261 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatgtaa gttagctcac
     1321 tcattaggca ccccaggctt tacactttat gcttccggct cgtatgttgt gtggaattgt
     1381 gagcggataa caatttcaca caggaaacag ctatgaccat gattacggat tcactggccg
     1441 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     1501 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     1561 aacagttgcg cagcctgaat ggcgaatggc gctttgcctg gtttccggca ccagaagcgg
     1621 tgccggaaag ctggctggag tgcgatcttc ctgaggccga tactgtcgtc gtcccctcaa
     1681 actggcagat gcacggttac gatgcgccca tctacaccaa cgtgacctat cccattacgg
     1741 tcaatccgcc gtttgttccc acggagaatc cgacgggttg ttactcgctc acatttaatg
     1801 ttgatgaaag ctggctacag gaaggccaga cgcgaattat ttttgatggc gttggaatta
     1861 gcttatcgac tgcacggtgc accaatgctt ctggcgtcag gcagccatcg gaagctgtgg
     1921 tatggctgtg caggtcgtaa atcactgcat aattcgtgtc gctcaaggcg cactcccgtt
     1981 ctggataatg ttttttgcgc cgacatcata acggttctgg caaatattct gaaatgagct
     2041 gttgacaatt aatcatcggc tcgtataatg tgtggaattg tgagcggata acaatttcac
     2101 acaggaaaca gtattcatgt cccctatact aggttattgg aaaattaagg gccttgtgca
     2161 acccactcga cttcttttgg aatatcttga agaaaaatat gaagagcatt tgtatgagcg
     2221 cgatgaaggt gataaatggc gaaacaaaaa gtttgaattg ggtttggagt ttcccaatct
     2281 tccttattat attgatggtg atgttaaatt aacacagtct atggccatca tacgttatat
     2341 agctgacaag cacaacatgt tgggtggttg tccaaaagag cgtgcagaga tttcaatgct
     2401 tgaaggagcg gttttggata ttagatacgg tgtttcgaga attgcatata gtaaagactt
     2461 tgaaactctc aaagttgatt ttcttagcaa gctacctgaa atgctgaaaa tgttcgaaga
     2521 tcgtttatgt cataaaacat atttaaatgg tgatcatgta acccatcctg acttcatgtt
     2581 gtatgacgct cttgatgttg ttttatacat ggacccaatg tgcctggatg cgttcccaaa
     2641 attagtttgt tttaaaaaac gtattgaagc tatcccacaa attgataagt acttgaaatc
     2701 cagcaagtat atagcatggc ctttgcaggg ctggcaagcc acgtttggtg gtggcgacca
     2761 tcctccaaaa tcggatctgg ttccgcgtgg atctatgtac ccatacgatg ttccagatta
     2821 cgcttaccca tacgatgttc cagattacgc ttacccatac gatgttccag attacgctgg
     2881 atccaagctt gaattcccgg gtcgactcga gcggccgcat cgtgactgac tgacgatctg
     2941 cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     3001 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     3061 tgttggcggg tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtataa
     3121 ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     3181 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     3241 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     3301 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     3361 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     3421 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     3481 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     3541 agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg
     3601 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     3661 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     3721 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     3781 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     3841 accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact
     3901 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     3961 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     4021 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     4081 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     4141 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     4201 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     4261 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     4321 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     4381 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     4441 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     4501 tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     4561 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     4621 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     4681 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     4741 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     4801 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     4861 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     4921 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     4981 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     5041 taaccgtatt accgcctttg ag
//
