LOCUS       sequence-334770-        3293 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  test plasmid for Golden Gate assembly, encodes fluorescent red 
            protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3293)
  AUTHORS   Matsumura I
  TITLE     Golden Gate assembly of BioBrick-compliant parts using Type II 
            restriction endonucleases.
  JOURNAL   Biotechniques. 2022 May;72(5):185-193. doi: 10.2144/btn-2021-0083. 
            Epub 2022 Mar 8.
  PUBMED    35255734
REFERENCE   2  (bases 1 to 3293)
  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"; doi: 
            "10.2144/btn-2021-0083"; journalName: "Biotechniques"; date: 
            "2022-05"; volume: "72"; issue: "5"; pages: "185-193"
COMMENT     Sequence Label: BsaI-tagRFP-BsaI-pUC-kan
FEATURES             Location/Qualifiers
     source          1..3293
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(168..756)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(248..267)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(934..1743)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     1657..1676
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1735..1754
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1885..1907)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2007..2026
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2220..2242
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     RBS             2288..2296
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(3059..3075)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3059..3075)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3072..3094)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3083..3099
                     /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(3107..3113,3114..3131,3132..3137))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3152..3173
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(join(3290..3293,1..14))
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
       61 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      121 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      181 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      241 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      301 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      361 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      421 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
      481 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      541 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      601 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
      661 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
      721 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
      781 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
      841 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aagcccaatc
      901 tgaataatgt tacaaccaat taaccaattc tgattagaaa aactcatcga gcatcaaatg
      961 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     1021 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     1081 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     1141 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     1201 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     1261 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     1321 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     1381 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     1441 tccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     1501 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     1561 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     1621 caagcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     1681 taaatcagca tccatgttgg aatttaatcg cggcctcgac gtttcccgtt gaatatggct
     1741 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat
     1801 atttttatct tgtgcaatgt aacatcagag attttgagac acgggccaga gctgcatcgc
     1861 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     1921 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     1981 cgggtgtcgg ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca
     2041 tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc
     2101 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     2161 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     2221 ccagtcacga cgttgtaaaa cgacggccag agaattcgag ctcggtacct cgcgaataca
     2281 tctagattaa ggaggtctca tatgtcagaa cttatcaagg aaaatatgca catgaaattg
     2341 tatatggaag gaactgtcaa taatcaccac tttaaatgta cctcagaagg agaaggaaaa
     2401 ccgtatgaag gtactcaaac catgcgtatt aaggttgtcg aaggtggacc acttcctttt
     2461 gcttttgata ttcttgcaac atcttttatg tacggatcac gtacctttat caatcataca
     2521 caaggtatcc cagatttctt taaacagtct tttcctgaag gatttacatg ggaacgtgtt
     2581 acaacttatg aagatggtgg agtcttgaca gctactcaag atacatcttt gcaagatggt
     2641 tgtttgatct acaatgtcaa gatccgtgga gtaaattttc catctaatgg tcctgttatg
     2701 cagaaaaaga ctcttggatg ggaagccaat accgaaatgt tgtatcctgc tgatggtgga
     2761 cttgagggtc gttcagatat ggcattgaaa cttgttggtg gaggtcattt gatctgtaat
     2821 tttaagacca cataccgtag taaaaagcca gcaaagaatc ttaagatgcc tggtgtatac
     2881 tacgtcgatc atcgtttgga acgtatcaaa gaagccgata aggaaactta tgttgaacaa
     2941 cacgaagtcg ccgtagcacg ttactgtgat ttgcctagta aattgggaca tggttgagac
     3001 catcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttggtg taatcatggt
     3061 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     3121 gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt
     3181 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     3241 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcg
//
