LOCUS       sequence-439379-        7436 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flag-tagged biotinylation enzyme for co-expression of Mapk8.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7436)
  TITLE     Ori lab plasmids
REFERENCE   2  (bases 1 to 7436)
  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"
COMMENT     Sequence Label: MAPK8_BirA_Cterm
FEATURES             Location/Qualifiers
     source          1..7436
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     181..198
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(239..262)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             complement(join(293..901,902..904,905..1255))
                     /codon_start=1
                     /product="promiscuous R118G mutant of the E. coli biotin 
                     protein ligase (Cronan, 2005)"
                     /label=BirA(R118G)
                     /note="also known as BirA* or BioID"
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     protein_bind    1266..1290
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2575..2599)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(2693..2717)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    2727..2745
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2748..2766
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     promoter        complement(2768..2971)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(2797..2817)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        2972..3351
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     3523..3542
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        3617..3721
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3722..3790,3791..4582)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3940..3959)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4753..5341
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5242..5261
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5495..5512
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5629..5650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5665..5670,5671..5688,5689..5695)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5703..5719
                     /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     5708..5730
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5727..5743
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5727..5743
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    5792..5913
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5803..5822)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5857..5876
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(6043..7065)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     protein_bind    complement(7073..7120)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     primer_bind     7252..7271
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    join(7404..7436,1..192)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
ORIGIN
        1 tattgtcttc ccaatcctcc cccttgctgt cctgccccac cccacccccc agaatagaat
       61 gacacctact cagacaatgc gatgcaattt cctcatttta ttaggaaagg acagtgggag
      121 tggcaccttc cagggtcaag gaaggcacgg gggaggggca aacaacagat ggctggcaac
      181 tagaaggcac agtcgaggct gatcagcggg tttaaacggg ccctctagac tcgagttatt
      241 tatcgtcatc gtctttgtag tctggcgcgc cgccgccggg gccagatccc cccttctcgg
      301 cgctccgcag gctgatctcg ccgcccatcc agggcttgat gatgccgtcc tgctccagca
      361 gcagggcgcc ctgcttgtcg atgccccggc tgatgccgaa gatctccttg tcgccgatga
      421 tcagcttcac gggccggttg atgaagttgt ccagcttctc ccaccggctc aggtaggggg
      481 ccaggccctc ctgctcgaac agctccaggg cggcccgcag ctcccggatc agcatggcgg
      541 ccagggtgtt ccggtccagg ttgatgccgg cctcctgcag ggtgatccag ccctggttca
      601 ccacgctctc ctccacccgc cgcatggcca tgttgatgcc ggcgccgatc acgatctggg
      661 cggcgtcgcc ggtcttgccg gtcagctcca ccaggatgcc ggccagcttc cggtcctgca
      721 ggtacaggtc gttgggccac ttcacccgca ccttgtcggc gcccagcttc cgcagcacct
      781 cggccatcac gatgccgatc accaggctca ggccgatggc ggcggcgggg ccctgctcca
      841 gccgccagaa catgctcagg tacaggttgg cgccgaaggg gctgaaccac ttccggcccc
      901 ggccgccccg gccggcctgc tggtactcgg cgatgcaggc gtcgccgctc ttcagctcgc
      961 cgatccggtc cagcaggtac tggttggtgc tgtcgatcac gggcagcacg gccacgctgc
     1021 cgccgtccag ctggcccagg atctgcttgg cgttcagcag ctggatgggc tcgggcaggc
     1081 tgtagccctt gccgggcacg gtgaacacgt ccacgcccca gtcccgcagg gtctggatgt
     1141 gcttgttgat ggcggcccgg ctcatgccca gggtctcgcc cagctgctcg ccgctgtgga
     1201 actcgccgtt ggccagcagg gcgatcagct tcaggggcac ggtgttgtcc ttcattctag
     1261 aatcaaccac tttgtacaag aaagttggca atctacagca gcccagaggc ccagctgctg
     1321 cttctagact gctgtctgta tcagaggcca aagtcggatc tgttgacatt gaagacacat
     1381 cattgacaga cgacgatgat gatggatgct gagagccatt gatcactgct gcacctaaag
     1441 gagagggctg cccccgtata actccattct tggttctctc ctccaagtcc ataacttcct
     1501 tatatatcaa ttctttccac tcttctattg tgtgttccct ttcatctaac tgcttgtcag
     1561 ggatctttgg tggtggagct tctgcttcag aaggatcata ccagacattg atgtacgggt
     1621 gttggagagc ttcatctaca gagatccttt tagatgcatc tattaccagc attttggata
     1681 acaaatccct tgcctgactg gctttaagtt tgttgtgttc tgagtcagct gggaaaagga
     1741 catcagggaa gagtttctca aagctatatc cagcatattt aggtctgttt tcaacgtaag
     1801 tccttactgt tggttgcagt ttcttcatga attcaggaca tggtgttcca agctgttcaa
     1861 taactttatt ccactgatca atatgatctg tacctgggaa caaaacacca cctttgatca
     1921 tttctcccat gatgcaccca actgaccaaa tgtcaacgtt ttccttgtag cccatgccaa
     1981 ggatgacctc gggtgctctg tagtagcgag tcactacata aggcgtcatc ataaaactcg
     2041 ttcctgcagt cctggccaga ccgaagtcaa gaatcttcaa agtgcaatca gattttacta
     2101 ctatattact gggctttaag tcccgatgaa taattccagc agaatgaagg tgcttgattc
     2161 cacacagcat ctgatagaga aggtaggaca ttctttcatg atctagctcc atctgaatca
     2221 cttggcaaag atttgcatcc atgagctcca tgactatgta aacatcttga aattcttcta
     2281 gggatttctg tggtgtgaaa acattcaaaa ggccaattat atttttgtga ttaacacatt
     2341 tcataagaac tagctctctg taggcccgct tggcatgagt ctgattctga aatggtcggc
     2401 ttagcttctt gattgcaaca tttctttcaa gaatggcatc ataagctgcg catactattc
     2461 cttgagctcc tgagcctata ggttttaaat tctgatatcg tttcaggact gtgaatgtag
     2521 aatctccaat ctctacacta taaaaattgt tgtcacgctt gcttctgctc atggtgccaa
     2581 cttttttgta caaacttgtc taaagcttaa gtttaaacgc tagagtccgg aggctggatc
     2641 ggtcccggtg tcttctatgg aggtcaaaac agcgtggatg gcgtctccag gcgatctgac
     2701 ggttcactaa acgagctcgt cgacgatctc tatcactgat agggagatct ctatcactga
     2761 tagggagagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     2821 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     2881 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     2941 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     3001 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc
     3061 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     3121 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     3181 gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc
     3241 agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa
     3301 ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt caacgcgtat
     3361 atctggcccg tacatcgcga agcagcgcaa aacgcctaac cctaagcaga ttcttcatgc
     3421 aattgtcggt caagccttgc cttgttgtag cttaaatttt gctcgcgcac tactcagcga
     3481 cctccaacac acaagcaggg agcagatact ggcttaacta tgcggcatca gagcagattg
     3541 tactgagagt gcaccatagg ggatcgggag atctcccgat ccgtcgacgt caggtggcac
     3601 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     3661 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     3721 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     3781 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     3841 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     3901 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     3961 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     4021 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     4081 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     4141 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     4201 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     4261 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     4321 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     4381 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     4441 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     4501 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     4561 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     4621 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     4681 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     4741 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     4801 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     4861 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     4921 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     4981 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     5041 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     5101 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     5161 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     5221 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     5281 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     5341 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     5401 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     5461 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     5521 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     5581 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     5641 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     5701 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     5761 ctagctagag gtcgacggta tacagacatg ataagataca ttgatgagtt tggacaaacc
     5821 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     5881 tttgtaacca ttataagctg caataaacaa gttggggtgg gcgaagaact ccagcatgag
     5941 atccccgcgc tggaggatca tccagccggc gtcccggaaa acgattccga agcccaacct
     6001 ttcatagaag gcggcggtgg aatcgaaatc tcgtagtacg tgctattcct ttgccctcgg
     6061 acgagtgctg gggcgtcggt ttccactatc ggcgagtact tctacacagc catcggtcca
     6121 gacggccgcg cttctgcggg cgatttgtgt acgcccgaca gtcccggctc cggatcggac
     6181 gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg aaattgccgt caaccaagct
     6241 ctgatagagt tggtcaagac caatgcggag catatacgcc cggagccgcg gcgatcctgc
     6301 aagctccgga tgcctccgct cgaagtagcg cgtctgctgc tccatacaag ccaaccacgg
     6361 cctccagaag aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg cctcgctcca
     6421 gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca ttgttggagc cgaaatccgc
     6481 gtgcacgagg tgccggactt cggggcagtc ctcggcccaa agcatcagct catcgagagc
     6541 ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt tgccagtgat acacatgggg
     6601 atcagcaatc gcgcatatga aatcacgcca tgtagtgtat tgaccgattc cttgcggtcc
     6661 gaatgggccg aacccgctcg tctggctaag atcggccgca gcgatcgcat ccatggcctc
     6721 cgcgaccggc tgcagaacag cgggcagttc ggtttcaggc aggtcttgca acgtgacacc
     6781 ctgtgcacgg cgggagatgc aataggtcag gctctcgctg aactccccaa tgtcaagcac
     6841 ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa acataacgat ctttgtagaa
     6901 accatcggcg cagctattta cccgcaggac atatccacgc cctcctacat cgaagctgaa
     6961 agcacgagat tcttcgccct ccgagagctg catcaggtcg gagacgctgt cgaacttttc
     7021 gatcagaaac ttctcgacag acgtcgcggt gagttcaggc tttttggcca aggaagttcc
     7081 tatactttct agagaatagg aacttcggaa taggaacttc taggtacgtg aaccatcacc
     7141 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
     7201 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
     7261 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
     7321 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtgg ggataccccc tagagcccca
     7381 gctggttctt tccgcctcag aagccataga gcccaccgca tccccagcat gcctgc
//
