LOCUS       sequence-439222-        7424 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flag-tagged biotinylation enzyme for co-expression of Mapk9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7424)
  TITLE     Ori lab plasmids
REFERENCE   2  (bases 1 to 7424)
  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: MAPK9_BirA_Cterm
FEATURES             Location/Qualifiers
     source          1..7424
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(619..643)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             join(654..1004,1005..1007,1008..1616)
                     /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             1647..1670
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1711..1728)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1717..1941
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2074..2093)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2225..2272
                     /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             2280..3302
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    3432..3553
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3469..3488)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3523..3542
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3602..3618)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3602..3618)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3615..3637)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3626..3642
                     /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(3650..3656,3657..3674,3675..3680))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3695..3716
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3833..3850)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4004..4592)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4084..4103)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4763..5554,5555..5623))
                     /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     5386..5405
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5624..5728)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5803..5822)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        5994..6373
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6374..6577
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6528..6548
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    6579..6597
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    6600..6618
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     6628..6652
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    6746..6770
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
ORIGIN
        1 tgaaaggttg tgtgatattc caaggcactg accatattga tcagtggaat aaagttattg
       61 agcagctggg aacaccatca gcagagttca tgaagaaact tcagccaact gtgaggaatt
      121 atgtcgaaaa cagaccaaag tatcctggaa tcaaatttga agaactcttt ccagattgga
      181 tattcccatc agaatctgag cgagacaaaa taaaaacaag tcaagccaga gatctgttat
      241 caaaaatgtt agtgattgat cctgacaagc ggatctctgt agacgaagct ctgcgtcacc
      301 catacatcac tgtttggtat gaccccgccg aagcagaagc cccaccacct caaatttatg
      361 atgcccagtt ggaagaaaga gaacatgcaa ttgaagaatg gaaagagcta atttacaaag
      421 aagtcatgga ttgggaagaa agaagcaaga atggtgttgt aaaagatcag ccttcagatg
      481 cagcagtaag tagcaacgcc actccttctc agtcttcatc gatcaatgac atttcatcca
      541 tgtccactga gcagacgctg gcctcagaca cagacagcag tcttgatgcc tcgacgggac
      601 cccttgaagg ctgtcgatgc ccaactttct tgtacaaagt ggttgattct agaatgaagg
      661 acaacaccgt gcccctgaag ctgatcgccc tgctggccaa cggcgagttc cacagcggcg
      721 agcagctggg cgagaccctg ggcatgagcc gggccgccat caacaagcac atccagaccc
      781 tgcgggactg gggcgtggac gtgttcaccg tgcccggcaa gggctacagc ctgcccgagc
      841 ccatccagct gctgaacgcc aagcagatcc tgggccagct ggacggcggc agcgtggccg
      901 tgctgcccgt gatcgacagc accaaccagt acctgctgga ccggatcggc gagctgaaga
      961 gcggcgacgc ctgcatcgcc gagtaccagc aggccggccg gggcggccgg ggccggaagt
     1021 ggttcagccc cttcggcgcc aacctgtacc tgagcatgtt ctggcggctg gagcagggcc
     1081 ccgccgccgc catcggcctg agcctggtga tcggcatcgt gatggccgag gtgctgcgga
     1141 agctgggcgc cgacaaggtg cgggtgaagt ggcccaacga cctgtacctg caggaccgga
     1201 agctggccgg catcctggtg gagctgaccg gcaagaccgg cgacgccgcc cagatcgtga
     1261 tcggcgccgg catcaacatg gccatgcggc gggtggagga gagcgtggtg aaccagggct
     1321 ggatcaccct gcaggaggcc ggcatcaacc tggaccggaa caccctggcc gccatgctga
     1381 tccgggagct gcgggccgcc ctggagctgt tcgagcagga gggcctggcc ccctacctga
     1441 gccggtggga gaagctggac aacttcatca accggcccgt gaagctgatc atcggcgaca
     1501 aggagatctt cggcatcagc cggggcatcg acaagcaggg cgccctgctg ctggagcagg
     1561 acggcatcat caagccctgg atgggcggcg agatcagcct gcggagcgcc gagaaggggg
     1621 gatctggccc cggcggcggc gcgccagact acaaagacga tgacgataaa taactcgagt
     1681 ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat
     1741 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     1801 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     1861 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg
     1921 gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt
     1981 atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     2041 tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc
     2101 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     2161 gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta
     2221 cctagaagtt cctattccga agttcctatt ctctagaaag tataggaact tccttggcca
     2281 aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag ttcgacagcg
     2341 tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc ttcgatgtag
     2401 gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac aaagatcgtt
     2461 atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt gacattgggg
     2521 agttcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc acgttgcaag
     2581 acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc atggatgcga
     2641 tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg caaggaatcg
     2701 gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat gtgtatcact
     2761 ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc gatgagctga
     2821 tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat ttcggctcca
     2881 acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc gaggcgatgt
     2941 tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg ttggcttgta
     3001 tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga tcgccgcggc
     3061 tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg gttgacggca
     3121 atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga tccggagccg
     3181 ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc gatggctgtg
     3241 tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg gcaaaggaat
     3301 agcacgtact acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     3361 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     3421 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3481 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3541 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     3601 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3661 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3721 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3781 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3841 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3901 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3961 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4021 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4081 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4141 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4201 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4261 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4321 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4381 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4441 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4501 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4561 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4621 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4681 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4741 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4801 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4861 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4921 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4981 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5041 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5101 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5161 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5221 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5281 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5341 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5401 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5461 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5521 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5581 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5641 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5701 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg
     5761 acggatcggg agatctcccg atcccctatg gtgcactctc agtacaatct gctctgatgc
     5821 cgcatagtta agccagtatc tgctccctgc ttgtgtgttg gaggtcgctg agtagtgcgc
     5881 gagcaaaatt taagctacaa caaggcaagg cttgaccgac aattgcatga agaatctgct
     5941 tagggttagg cgttttgcgc tgcttcgcga tgtacgggcc agatatacgc gttgacattg
     6001 attattgact agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat
     6061 ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     6121 ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag ggactttcca
     6181 ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac atcaagtgta
     6241 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     6301 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     6361 cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat agcggtttga
     6421 ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt tttggcacca
     6481 aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc aaatgggcgg
     6541 taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg atagagatct
     6601 ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga tcgcctggag
     6661 acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca gcctccggac
     6721 tctagcgttt aaacttaagc tttagacaag tttgtacaaa aaagttggca tgagcgacag
     6781 taaatgtgac agtcagtttt atagtgtgca agtggcagac tcaaccttca ctgtcctaaa
     6841 acgttaccag cagctgaaac caattggctc tggggcccaa gggattgttt gtgctgcatt
     6901 tgatacagtt cttgggataa atgttgcagt caagaaacta agccgtcctt ttcagaacca
     6961 aactcatgca aagagagctt atcgtgaact tgtcctctta aaatgtgtca atcataaaaa
     7021 tataattagt ttgttaaatg tgtttacacc acaaaaaact ctagaagaat ttcaagatgt
     7081 gtatttggtt atggaattaa tggatgctaa cttatgtcag gttattcaca tggagctgga
     7141 tcatgaaaga atgtcctacc ttctttacca gatgctttgt ggtattaaac atctgcattc
     7201 agctggtata attcatagag atttgaagcc tagcaacatt gttgtgaaat cagactgcac
     7261 cctgaagatc cttgactttg gcctggcccg gacagcgtgc actaacttca tgatgacccc
     7321 ttacgtggtg acacggtact accgggcgcc cgaagtcatc ctgggtatgg gctacaaaga
     7381 gaacgttgat atctggtcag tgggttgcat catgggagag ctgg
//
