LOCUS       sequence-248755-        5394 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient expression of biotin ligase BirA and p19 in plant 
            protoplast.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5394)
  AUTHORS   Tu X, Mejia-Guerra MK, Valdes Franco JA, Tzeng D, Chu PY, Shen W, 
            Wei Y, Dai X, Li P, Buckler ES, Zhong S
  TITLE     Reconstructing the maize leaf regulatory network using ChIP-seq data
            of 104 transcription factors.
  JOURNAL   Nat Commun. 2020 Oct 9;11(1):5089. doi: 10.1038/s41467-020-18832-8.
  PUBMED    33037196
REFERENCE   2  (bases 1 to 5394)
  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: "2020-10-9"; volume: "11(1):5089. doi"; pages: " 
            10.1038/s41467-020-18832-8"
COMMENT     Sequence Label: pBirA-P19
FEATURES             Location/Qualifiers
     source          1..5394
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        547..593
                     /label=minimal CaMV 35S promoter
                     /note="minimal 35S promoter from cauliflower mosaic virus"
     CDS             649..1611
                     /codon_start=1
                     /product="E. coli biotin protein ligase"
                     /label=BirA
                     /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD
                     WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG
                     DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK
                     LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG
                     WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG
                     DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK"
     primer_bind     complement(1893..1909)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1893..1909)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1906..1928)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1917..1933
                     /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(1941..1947,1948..1965,1966..1971))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1986..2007
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2124..2141)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2295..2883)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2375..2394)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3054..3845,3846..3914))
                     /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     3677..3696
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3915..4019)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4087..4105
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4143..4165)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4265..4284
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     polyA_signal    4305..4481
                     /label=CaMV poly(A) signal
                     /note="cauliflower mosaic virus polyadenylation signal"
     promoter        complement(join(5108..5394,1..59))
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
ORIGIN
        1 tagctgggca atggaatccg aggaggtttc ccgaaattac cctttgttga aaagtctcaa
       61 tagccctttg gtcttctgag actgtatctt tgacattttt ggagtagggg ggtaccgagc
      121 tcgaattctt aatcgcaacc tgcagcatat aactactgta tgtgctatgg tatggactat
      181 ggaatatgat taaagataag atgggctcat agagtaaaac gaggcgaggg acctataaac
      241 ctcccttctg agacttttca acaaagggta atatccggaa acctcctcgg attccattgc
      301 ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc
      361 catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa
      421 gatggacccc caccccacga ggagcatcgt ggaaaaagaa gacgttccaa ccacgtcttc
      481 aaagcaagtg gattgatgtg atatctccac tgacgtaagg gatgacgcac aatcccacta
      541 tccttcgcaa gacccttcct ctatataagg aagttcattt catttggaga ggaagatcaa
      601 atcagcagga aaatcaagaa ccaaaagtct cccgaaaagc aacgaacaat gaaagataat
      661 acagttcccc tcaaacttat cgcacttctg gcaaacgggg aatttcactc tggagaacaa
      721 cttggagaga ctttgggtat gtcacgggcg gcaatcaaca aacatataca gactttgcgg
      781 gactggggag tggacgtgtt tacagttcct ggtaaaggat atagcctgcc cgagccaata
      841 caacttctga acgcgaaaca gattctgggt cagttggacg gcggtagcgt tgccgtcttg
      901 cctgtgatag attcaaccaa ccagtatctt ctcgacagga taggagaatt gaaatcgggt
      961 gacgcctgta tcgctgaata tcaacaggca ggccgcggtc ggaggggtag aaagtggttc
     1021 tccccctttg gggccaatct ttacctgtcc atgttctggc ggttggaaca ggggccggca
     1081 gcggcaatcg gactttccct tgtcataggg atagtcatgg ctgaagtcct ccgcaagttg
     1141 ggcgcggaca aggtcagagt gaagtggccg aacgacctct acttgcagga cagaaaactg
     1201 gccggcattt tggtggaact tacgggcaag acgggcgacg ccgctcagat tgttattgga
     1261 gctggtatta atatggcgat gagaagagtg gaagaatctg ttgttaatca aggttggatt
     1321 accttgcagg aagctggcat aaacttggat agaaatacgt tggctgctat gttgattaga
     1381 gaattgcggg cagcgttgga actcttcgaa caggaaggac tggcacctta tttgtcgagg
     1441 tgggagaagc tcgacaactt cattaacagg ccggttaagc tgataattgg ggacaaggaa
     1501 attttcggta tcagcagggg tatcgacaaa cagggtgccc tgctccttga acaggacgga
     1561 attatcaagc cgtggatggg tggagagatt tctctcagat ccgcagaaaa atagcggcca
     1621 tgctagagtc cgcaaaaatc accagtctct ctctacaaat ctatctctct ctatttttct
     1681 ccagaataat gtgtgagtag ttcccagata agggaattag ggttcttata gggtttcgct
     1741 catgtgttga gcatataaga aacccttagt atgtatttgt atttgtaaaa tacttctatc
     1801 aataaaattt ctaattccta aaaccaaaat ccagtgacct gtcggactac acaatcgtcg
     1861 aacggcaggc gtgcaaactt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     1921 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     1981 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     2041 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     2101 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     2161 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     2221 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     2281 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     2341 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     2401 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     2461 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     2521 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     2581 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     2641 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     2701 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     2761 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     2821 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     2881 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     2941 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     3001 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     3061 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     3121 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     3181 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     3241 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     3301 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     3361 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     3421 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     3481 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     3541 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     3601 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     3661 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     3721 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     3781 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     3841 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     3901 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     3961 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatggg ggttccgcgc
     4021 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     4081 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     4141 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     4201 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     4261 tatgcggcat cagagcagat tgtactgaga gtgcaccata tggagatctg gattttagta
     4321 ctggattttg gttttaggaa ttagaaattt tattgataga agtattttac aaatacaaat
     4381 acatactaag ggtttcttat atgctcaaca catgagcgaa accctataag aaccctaatt
     4441 tcccttatcg ggaaactact cacacattat ttatggagaa aatagagaga gatagatttg
     4501 tagagagaga ctggtgattt cagcgtaccg aattcgagct cgccctcgac ttactcgctt
     4561 tctttttcga aggtttgagt accttcaggg catcctcttg atacattact ttccacttcg
     4621 attggggcaa gctgtagcag ttcttgctta gaccgaattg ccatctcaca gagatgctga
     4681 agagttcgag accctccaga aacggtgata ctaactcctc gaaaccgaat actataggta
     4741 catccgatct ggtcgaaacc gaaaaatcga gatgctgcat agttaaccga atctcccgtc
     4801 caagatccaa ggactctgtg cagtgaagct tccgtcctgt cgtatctgag atatctctta
     4861 aatacaactt tcccgaaacc ccagctttcc ttgaaaccaa ggggattatc ttgattcgaa
     4921 ttcgtctcat cgttatgtag ccgccactca gtccaactcg gactttcgtc aggaagtttg
     4981 aagggagaag tggtacctcc tgatcctcca tcccaacgtt cactgttagc ttgttcccta
     5041 gcgtcgtttc cttgtatagc tcgttccata tcgatttaag gggatcctct agagtcgaag
     5101 cttgggctgt cctctccaaa tgaaatgaac ttccttatat agaggaaggg tcttgcgaag
     5161 gatagtggga ttgtgcgtca tcccttacgt cagtggagat gtcacatcaa tccacttgct
     5221 ttgaagacgt ggttggaacg tcttcttttt ccacgatgct cctcgtgggt gggggtccat
     5281 ctttgggacc actgtcggca gaggcatctt gaatgatagc ctttccttta tcgcaatgat
     5341 ggcatttgta ggagccacct tccttttcta ctgtcctttc gatgaagtga caga
//
