LOCUS       sequence-312901-        3022 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Level 0.5 Golden Gate backbone, containing loxP sites..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3022)
  AUTHORS   Harrington SA, Backhaus AE, Fox S, Rogers C, Borrill P, Uauy C, 
            Richardson A
  TITLE     A heat-shock inducible system for flexible gene expression in 
            cereals.
  JOURNAL   Plant Methods. 2020 Oct 14;16:137. doi: 10.1186/s13007-020-00677-3. 
            eCollection 2020.
  PUBMED    33072173
REFERENCE   2  (bases 1 to 3022)
  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: "Plant 
            Methods."; date: "2020-10-14"; volume: "16:137. doi"; pages: " 
            10.1186/s13007-020-00677-3. eCollection 2020"
COMMENT     Sequence Label: EC10161
FEATURES             Location/Qualifiers
     source          1..3022
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     301..317
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     328..347
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        328..346
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             351..362
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(406..439)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(551..874)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     556..575
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     784..800
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    801..857
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(870..886)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(883..905)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    894..910
                     /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(918..924,925..942,943..948))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    963..984
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(1057..1090)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(1236..1253)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1346..1934)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1426..1445)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2108..2902)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     promoter        complement(2903..3007)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
       61 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      121 tagaccgaga tagggttgag tggccgctac agggcgctcc cattcgccat tcaggctgcg
      181 caactgttgg gaagggcgtt tcggtgcggg cctcttcgct attacgccag ctggcgaaag
      241 ggggatgtgc tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt
      301 gtaaaacgac ggccagtgag cgcgacgtaa tacgactcac tatagggcga attgaaggaa
      361 ggccgtcaag gccgcatcac ttcgtgcacg tctcatactg acctaataac ttcgtatagc
      421 atacattata cgaagttata ttaagggttg aatgtgagac cgtcacagct tgtctgtaag
      481 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
      541 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
      601 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccattcg ccattcaggc
      661 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      721 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      781 gttgtaaaac gacggccagt gaattcgagc tcggtacccg gggatcctct agagtcgacc
      841 tgcaggcatg caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
      901 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
      961 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggca
     1021 aacctgtcgt gccagctgcg gtctcacgct gacctaataa cttcgtatag catacattat
     1081 acgaagttat attaagggtt gaatgtgaga cgcacagagt gctgggcctc atgggccttc
     1141 ctttcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta acatggtcat
     1201 agctgtttcc ttgcgtattg ggcgctctcc gcttcctcgc tcactgactc gctgcgctcg
     1261 gtcgttcggg taaagcctgg ggtgcctaat gagcaaaagg ccagcaaaag gccaggaacc
     1321 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1381 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1441 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1501 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1561 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1621 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1681 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1741 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     1801 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1861 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1921 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1981 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     2041 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     2101 acagttatta gaaaaattca tccagcagac gataaaacgc aatacgctgg ctatccggtg
     2161 ccgcaatgcc atacagcacc agaaaacgat ccgcccattc gccgcccagt tcttccgcaa
     2221 tatcacgggt ggccagcgca atatcctgat aacgatccgc cacgcccaga cggccgcaat
     2281 caataaagcc gctaaaacgg ccattttcca ccataatgtt cggcaggcac gcatcaccat
     2341 gggtcaccac cagatcttcg ccatccggca tgctcgcttt cagacgcgca aacagctctg
     2401 ccggtgccag gccctgatgt tcttcatcca gatcatcctg atccaccagg cccgcttcca
     2461 tacgggtacg cgcacgttca atacgatgtt tcgcctgatg atcaaacgga caggtcgccg
     2521 ggtccagggt atgcagacga cgcatggcat ccgccataat gctcactttt tctgccggcg
     2581 ccagatggct agacagcaga tcctgacccg gcacttcgcc cagcagcagc caatcacggc
     2641 ccgcttcggt caccacatcc agcaccgccg cacacggaac accggtggtg gccagccagc
     2701 tcagacgcgc cgcttcatcc tgcagctcgt tcagcgcacc gctcagatcg gttttcacaa
     2761 acagcaccgg acgaccctgc gcgctcagac gaaacaccgc cgcatcagag cagccaatgg
     2821 tctgctgcgc ccaatcatag ccaaacagac gttccaccca cgctgccggg ctacccgcat
     2881 gcaggccatc ctgttcaatc atactcttcc tttttcaata ttattgaagc atttatcagg
     2941 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     3001 ttccgcgcac atttccccga aa
//
