LOCUS       sequence-434331-        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     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    820..838
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    841..859
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     869..893
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    987..1011
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(2296..2320)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(2331..2681,2682..2684,2685..3293)
                     /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"
     CDS             3324..3347
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3388..3405)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3394..3618
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3751..3770)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3902..3949
                     /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)."
     CDS             3957..4979
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGDFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5109..5230
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5146..5165)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5200..5219
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5279..5295)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5279..5295)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5292..5314)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5303..5319
                     /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)."
     promoter        complement(join(5327..5333,5334..5351,5352..5357))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5372..5393
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5510..5527)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5681..6269)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5761..5780)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6440..7231,7232..7300))
                     /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     7063..7082
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7301..7405)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ccctatcagt gatagagatc
      841 tccctatcag tgatagagat cgtcgacgag ctcgtttagt gaaccgtcag atcgcctgga
      901 gacgccatcc acgctgtttt gacctccata gaagacaccg ggaccgatcc agcctccgga
      961 ctctagcgtt taaacttaag ctttagacaa gtttgtacaa aaaagttggc accatgagca
     1021 gaagcaagcg tgacaacaat ttttatagtg tagagattgg agattctaca ttcacagtcc
     1081 tgaaacgata tcagaattta aaacctatag gctcaggagc tcaaggaata gtatgcgcag
     1141 cttatgatgc cattcttgaa agaaatgttg caatcaagaa gctaagccga ccatttcaga
     1201 atcagactca tgccaagcgg gcctacagag agctagttct tatgaaatgt gttaatcaca
     1261 aaaatataat tggccttttg aatgttttca caccacagaa atccctagaa gaatttcaag
     1321 atgtttacat agtcatggag ctcatggatg caaatctttg ccaagtgatt cagatggagc
     1381 tagatcatga aagaatgtcc taccttctct atcagatgct gtgtggaatc aagcaccttc
     1441 attctgctgg aattattcat cgggacttaa agcccagtaa tatagtagta aaatctgatt
     1501 gcactttgaa gattcttgac ttcggtctgg ccaggactgc aggaacgagt tttatgatga
     1561 cgccttatgt agtgactcgc tactacagag cacccgaggt catccttggc atgggctaca
     1621 aggaaaacgt tgacatttgg tcagttgggt gcatcatggg agaaatgatc aaaggtggtg
     1681 ttttgttccc aggtacagat catattgatc agtggaataa agttattgaa cagcttggaa
     1741 caccatgtcc tgaattcatg aagaaactgc aaccaacagt aaggacttac gttgaaaaca
     1801 gacctaaata tgctggatat agctttgaga aactcttccc tgatgtcctt ttcccagctg
     1861 actcagaaca caacaaactt aaagccagtc aggcaaggga tttgttatcc aaaatgctgg
     1921 taatagatgc atctaaaagg atctctgtag atgaagctct ccaacacccg tacatcaatg
     1981 tctggtatga tccttctgaa gcagaagctc caccaccaaa gatccctgac aagcagttag
     2041 atgaaaggga acacacaata gaagagtgga aagaattgat atataaggaa gttatggact
     2101 tggaggagag aaccaagaat ggagttatac gggggcagcc ctctccttta ggtgcagcag
     2161 tgatcaatgg ctctcagcat ccatcatcat cgtcgtctgt caatgatgtg tcttcaatgt
     2221 caacagatcc gactttggcc tctgatacag acagcagtct agaagcagca gctgggcctc
     2281 tgggctgctg tagattgcca actttcttgt acaaagtggt tgattctaga atgaaggaca
     2341 acaccgtgcc cctgaagctg atcgccctgc tggccaacgg cgagttccac agcggcgagc
     2401 agctgggcga gaccctgggc atgagccggg ccgccatcaa caagcacatc cagaccctgc
     2461 gggactgggg cgtggacgtg ttcaccgtgc ccggcaaggg ctacagcctg cccgagccca
     2521 tccagctgct gaacgccaag cagatcctgg gccagctgga cggcggcagc gtggccgtgc
     2581 tgcccgtgat cgacagcacc aaccagtacc tgctggaccg gatcggcgag ctgaagagcg
     2641 gcgacgcctg catcgccgag taccagcagg ccggccgggg cggccggggc cggaagtggt
     2701 tcagcccctt cggcgccaac ctgtacctga gcatgttctg gcggctggag cagggccccg
     2761 ccgccgccat cggcctgagc ctggtgatcg gcatcgtgat ggccgaggtg ctgcggaagc
     2821 tgggcgccga caaggtgcgg gtgaagtggc ccaacgacct gtacctgcag gaccggaagc
     2881 tggccggcat cctggtggag ctgaccggca agaccggcga cgccgcccag atcgtgatcg
     2941 gcgccggcat caacatggcc atgcggcggg tggaggagag cgtggtgaac cagggctgga
     3001 tcaccctgca ggaggccggc atcaacctgg accggaacac cctggccgcc atgctgatcc
     3061 gggagctgcg ggccgccctg gagctgttcg agcaggaggg cctggccccc tacctgagcc
     3121 ggtgggagaa gctggacaac ttcatcaacc ggcccgtgaa gctgatcatc ggcgacaagg
     3181 agatcttcgg catcagccgg ggcatcgaca agcagggcgc cctgctgctg gagcaggacg
     3241 gcatcatcaa gccctggatg ggcggcgaga tcagcctgcg gagcgccgag aaggggggat
     3301 ctggccccgg cggcggcgcg ccagactaca aagacgatga cgataaataa ctcgagtcta
     3361 gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     3421 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3481 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3541 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3601 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc
     3661 cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
     3721 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
     3781 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
     3841 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtacct
     3901 agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc ttggccaaaa
     3961 agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc gacagcgtct
     4021 ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc gatgtaggag
     4081 ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa gatcgttatg
     4141 tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac attggggatt
     4201 tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc
     4261 tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg gatgcgatcg
     4321 ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc
     4381 aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg tatcactggc
     4441 aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc
     4501 tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc ggctccaaca
     4561 atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag gcgatgttcg
     4621 gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg gcttgtatgg
     4681 agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg ccgcggctcc
     4741 gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt gacggcaatt
     4801 tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc ggagccggga
     4861 ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag
     4921 aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca aaggaatagc
     4981 acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc ttcggaatcg
     5041 ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg gagttcttcg
     5101 cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5161 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5221 atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg taatcatggt
     5281 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     5341 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     5401 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     5461 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg
     5521 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5581 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5641 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5701 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5761 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5821 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5881 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5941 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6001 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6061 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6121 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6181 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6241 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6301 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6361 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6421 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6481 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6541 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     6601 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6661 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6721 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     6781 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6841 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6901 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     6961 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7021 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7081 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7141 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7201 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7261 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7321 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7381 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtc
//
