LOCUS       sequence-298546-        3103 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 3103)
  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 3103)
  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..3103
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     293..309
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     320..339
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        320..338
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             343..354
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(438..471)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(583..906)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     588..607
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     816..832
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    833..889
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(902..918)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(915..937)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    926..942
                     /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(950..956,957..974,975..980))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    995..1016
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     protein_bind    complement(1089..1122)
                     /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(1309..1326)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1419..2007)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1499..1518)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2181..2975)
                     /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(2976..3080)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtggccgct acagggcgct cccattcgcc attcaggctg cgcaactgtt
      181 gggaagggcg tttcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
      241 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
      301 acggccagtg agcgcgacgt aatacgactc actatagggc gaattgaagg aaggccgtca
      361 aggcctaggc gcgccatgag ctctaaagct tcgctcgagc tgcggccgcc acttcgtgca
      421 cgtctcatac tgacctaata acttcgtata gcatacatta tacgaagtta tattaagggt
      481 tgaatgtgag accgtcacag cttgtctgta agcggatgcc gggagcagac aagcccgtca
      541 gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt aactatgcgg catcagagca
      601 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa
      661 ataccgcatc aggcgccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
      721 gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag
      781 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattcga
      841 gctcggtacc cggggatcct ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat
      901 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
      961 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     1021 cgttgcgctc actgcccgct ttccagtcgg caaacctgtc gtgccagctg cggtctcacg
     1081 ctgacctaat aacttcgtat agcatacatt atacgaagtt atattaaggg ttgaatgtga
     1141 gacgcacaga gtggcggccg cttggatccc agaattctag gtacctctta attaactggc
     1201 ctcatgggcc ttcctttcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1261 ttaacatggt catagctgtt tccttgcgta ttgggcgctc tccgcttcct cgctcactga
     1321 ctcgctgcgc tcggtcgttc gggtaaagcc tggggtgcct aatgagcaaa aggccagcaa
     1381 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     1441 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     1501 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     1561 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     1621 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     1681 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     1741 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     1801 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     1861 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     1921 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     1981 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     2041 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     2101 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     2161 taaacttggt ctgacagtta ttagaaaaat tcatccagca gacgataaaa cgcaatacgc
     2221 tggctatccg gtgccgcaat gccatacagc accagaaaac gatccgccca ttcgccgccc
     2281 agttcttccg caatatcacg ggtggccagc gcaatatcct gataacgatc cgccacgccc
     2341 agacggccgc aatcaataaa gccgctaaaa cggccatttt ccaccataat gttcggcagg
     2401 cacgcatcac catgggtcac caccagatct tcgccatccg gcatgctcgc tttcagacgc
     2461 gcaaacagct ctgccggtgc caggccctga tgttcttcat ccagatcatc ctgatccacc
     2521 aggcccgctt ccatacgggt acgcgcacgt tcaatacgat gtttcgcctg atgatcaaac
     2581 ggacaggtcg ccgggtccag ggtatgcaga cgacgcatgg catccgccat aatgctcact
     2641 ttttctgccg gcgccagatg gctagacagc agatcctgac ccggcacttc gcccagcagc
     2701 agccaatcac ggcccgcttc ggtcaccaca tccagcaccg ccgcacacgg aacaccggtg
     2761 gtggccagcc agctcagacg cgccgcttca tcctgcagct cgttcagcgc accgctcaga
     2821 tcggttttca caaacagcac cggacgaccc tgcgcgctca gacgaaacac cgccgcatca
     2881 gagcagccaa tggtctgctg cgcccaatca tagccaaaca gacgttccac ccacgctgcc
     2941 gggctacccg catgcaggcc atcctgttca atcatactct tcctttttca atattattga
     3001 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3061 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cac
//
