LOCUS       sequence-192792-        7367 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of GFP-AURKA for protein purification in bacteria.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7367)
  AUTHORS   Bertolin G, Sizaire F, Herbomel G, Reboutier D, Prigent C, Tramier M
  TITLE     A FRET biosensor reveals spatiotemporal activation and functions of 
            aurora kinase A in living cells.
  JOURNAL   Nat Commun. 2016 Sep 14;7:12674. doi: 10.1038/ncomms12674.
  PUBMED    27624869
REFERENCE   2  (bases 1 to 7367)
  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: "Nat 
            Commun."; date: "2016-09-14"; volume: "7:12674. doi"; pages: " 
            10.1038/ncomms12674"
COMMENT     Sequence Label: pET21-GFP-AURKA
FEATURES             Location/Qualifiers
     source          1..7367
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     177..196
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     280..299
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        280..298
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    299..323
                     /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             338..360
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             368..400
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             join(410..412,413..415,416..1126)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(410..412,413..415,416..1126)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(455..476)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(716..735)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1063..1084
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2042..2053
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     CDS             2378..2395
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2448..2466)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2462..2509
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2546..3001
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2633..2652)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2843..2864
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3027..3131
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3132..3200,3201..3992)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3350..3369)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4163..4751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4652..4671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4905..4922
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4937..5076
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5002..5021)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5162..5184
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5178..5369)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6178..7260)
                     /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     7222..7241
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7261..7338)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg
       61 aatggtgcat gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata
      121 cccacgccga aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg
      181 atgtcggcga tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg
      241 cgtccggcgt agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg
      301 aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga
      361 tatacatatg gctagcatga ctggtggaca gcaaatgggt cgcggatcca tggtgagcaa
      421 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
      481 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac
      541 cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac
      601 cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt
      661 cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga
      721 cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat
      781 cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta
      841 caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt
      901 gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca
      961 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac
     1021 ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     1081 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaaggaat tcgagctcac
     1141 catggaccga tctaaagaaa actgcatttc aggacctgtt aaggctacag ctccagttgg
     1201 aggtccaaaa cgtgttctcg tgactcagca atttccttgt cagaatccat tacctgtaaa
     1261 tagtggccag gctcagcggg tcttgtgtcc ttcaaattct tcccagcgcg ttcctttgca
     1321 agcacaaaaa cttgtctcca gtcacaagcc ggttcagaat cagaagcaga agcaattgca
     1381 ggcaaccagt gtacctcatc ctgtctccag gccactgaat aacacccaaa agagcaagca
     1441 gcccctgcca tcggcacctg aaaataatcc tgaggaggaa ctggcatcaa aacagaaaaa
     1501 tgaagaatca aaaaagaggc agtgggcttt ggaagacttt gaaattggtc gccctctggg
     1561 taaaggaaag tttggtaatg tttatttggc aagagaaaag caaagcaagt ttattctggc
     1621 tcttaaagtg ttatttaaag ctcagctgga gaaagccgga gtggagcatc agctcagaag
     1681 agaagtagaa atacagtccc accttcggca tcctaatatt cttagactgt atggttattt
     1741 ccatgatgct accagagtct acctaattct ggaatatgca ccacttggaa cagtttatag
     1801 agaacttcag aaactttcaa agtttgatga gcagagaact gctacttata taacagaatt
     1861 ggcaaatgcc ctgtcttact gtcattcgaa gagagttatt catagagaca ttaagccaga
     1921 gaacttactt cttggatcag ctggagagct taaaattgca gattttgggt ggtcagtaca
     1981 tgctccatct tccaggagga ccactctctg tggcaccctg gactacctgc cccctgaaat
     2041 gattgaaggt cggatgcatg atgagaaggt ggatctctgg agccttggag ttctttgcta
     2101 tgaattttta gttgggaagc ctccttttga ggcaaacaca taccaagaga cctacaaaag
     2161 aatatcacgg gttgagttca cattccctga ctttgtaaca gagggagcca gggacctcat
     2221 ttcaagactg ttgaagcata atcccagcca gaggccaatg ctcagagaag tacttgaaca
     2281 cccctggatc acagcaaatt catcaaaacc atcaaattgc caaaacaaag aatcagctag
     2341 caaacagtct gtcgacaagc ttgcggccgc actcgagcac caccaccacc accactgaga
     2401 tccggctgct aacaaagccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     2461 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     2521 aactatatcc ggattggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2581 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2641 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2701 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2761 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2821 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2881 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2941 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt
     3001 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3061 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3121 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     3181 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     3241 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     3301 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     3361 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     3421 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     3481 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     3541 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     3601 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     3661 acaccacgat gcctgcagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     3721 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     3781 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     3841 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     3901 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     3961 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     4021 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     4081 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     4141 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4201 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4261 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     4321 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4381 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4441 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4501 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4561 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4621 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4681 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4741 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4801 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     4861 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     4921 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     4981 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     5041 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     5101 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     5161 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     5221 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     5281 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     5341 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     5401 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     5461 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     5521 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     5581 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     5641 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     5701 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     5761 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     5821 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     5881 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     5941 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     6001 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     6061 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     6121 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     6181 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6241 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     6301 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     6361 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     6421 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     6481 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     6541 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     6601 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     6661 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     6721 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     6781 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     6841 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     6901 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     6961 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     7021 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     7081 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     7141 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     7201 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     7261 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     7321 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgac
//
