LOCUS       sequence-256099-        5053 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Empty backbone for FLAG tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5053)
  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 5053)
  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-1-3xFLAG
FEATURES             Location/Qualifiers
     source          1..5053
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     46..63
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        136..213
                     /gene="lacI"
                     /label=lacI promoter
     CDS             214..1296
                     /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(233..252)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(1345..1350,1351..1368,1369..1375)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1383..1399
                     /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     1407..1423
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1407..1423
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             1419..1946
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(1439..1456)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1439..1455)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1448..1470)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     promoter        join(2048..2053,2054..2069,2070..2076)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    2084..2100
                     /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             2123..2776
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     2734..2756
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             2783..2800
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2804..2875
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     primer_bind     complement(2968..2990)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3135..3153)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3221..3325
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3326..3394,3395..4186)
                     /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(3544..3563)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4357..4945
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4846..4865
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
       61 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
      121 caccgcataa attccgacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag
      181 cgcccggaag agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc
      241 gcagagtatg ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac
      301 gtttctgcga aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc
      361 aaccgcgtgg cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc
      421 agtctggccc tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa
      481 ctgggtgcca gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg
      541 gcggtgcaca atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat
      601 gaccaggatg ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat
      661 gtctctgacc agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg
      721 ggcgtggagc atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta
      781 agttctgtct cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa
      841 attcagccga tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc
      901 atgcaaatgc tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg
      961 gcgctgggcg caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg
     1021 gtagtgggat acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc
     1081 aaacaggatt ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag
     1141 ggccaggcgg tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc
     1201 ctggcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     1261 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta
     1321 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     1381 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acggattcac
     1441 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     1501 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     1561 cttcccaaca gttgcgcagc ctgaatggcg aatggcgctt tgcctggttt ccggcaccag
     1621 aagcggtgcc ggaaagctgg ctggagtgcg atcttcctga ggccgatact gtcgtcgtcc
     1681 cctcaaactg gcagatgcac ggttacgatg cgcccatcta caccaacgtg acctatccca
     1741 ttacggtcaa tccgccgttt gttcccacgg agaatccgac gggttgttac tcgctcacat
     1801 ttaatgttga tgaaagctgg ctacaggaag gccagacgcg aattattttt gatggcgttg
     1861 gaattagctt atcgactgca cggtgcacca atgcttctgg cgtcaggcag ccatcggaag
     1921 ctgtggtatg gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact
     1981 cccgttctgg ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa
     2041 tgagctgttg acaattaatc atcggctcgt ataatgtgtg gaattgtgag cggataacaa
     2101 tttcacacag gaaacagtat tcatgtcccc tatactaggt tattggaaaa ttaagggcct
     2161 tgtgcaaccc actcgacttc ttttggaata tcttgaagaa aaatatgaag agcatttgta
     2221 tgagcgcgat gaaggtgata aatggcgaaa caaaaagttt gaattgggtt tggagtttcc
     2281 caatcttcct tattatattg atggtgatgt taaattaaca cagtctatgg ccatcatacg
     2341 ttatatagct gacaagcaca acatgttggg tggttgtcca aaagagcgtg cagagatttc
     2401 aatgcttgaa ggagcggttt tggatattag atacggtgtt tcgagaattg catatagtaa
     2461 agactttgaa actctcaaag ttgattttct tagcaagcta cctgaaatgc tgaaaatgtt
     2521 cgaagatcgt ttatgtcata aaacatattt aaatggtgat catgtaaccc atcctgactt
     2581 catgttgtat gacgctcttg atgttgtttt atacatggac ccaatgtgcc tggatgcgtt
     2641 cccaaaatta gtttgtttta aaaaacgtat tgaagctatc ccacaaattg ataagtactt
     2701 gaaatccagc aagtatatag catggccttt gcagggctgg caagccacgt ttggtggtgg
     2761 cgaccatcct ccaaaatcgg atctggttcc gcgtggatct atggattaca aggatgacga
     2821 cgataaggat tacaaggatg acgacgataa ggattacaag gatgacgacg ataagggatc
     2881 caagcttgaa ttcccgggtc gactcgagcg gccgcatcgt gactgactga cgatctgcct
     2941 cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac
     3001 agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt
     3061 tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtataattc
     3121 ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat
     3181 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     3241 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     3301 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     3361 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     3421 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     3481 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     3541 tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg caagagcaac tcggtcgccg
     3601 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3661 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3721 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3781 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3841 aaacgacgag cgtgacacca cgatgcctgc agcaatggca acaacgttgc gcaaactatt
     3901 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3961 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     4021 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     4081 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     4141 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     4201 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     4261 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     4321 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     4381 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     4441 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     4501 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     4561 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     4621 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4681 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4741 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4801 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4861 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4921 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4981 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     5041 ccgtattacc gcc
//
