LOCUS       sequence-492590-        3426 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Minicircle producer plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3426)
  AUTHORS   Migliori V, Bruntraeger MB, Gyulev IS, Lichou F, Burgold T, 
            Gitterman DP, Iwama S, Trinh AL, Washer SJ, Jones CP, Trynka G, 
            Bassett AR
  TITLE     ONE-STEP tagging: a versatile method for rapid site-specific 
            integration by simultaneous reagent delivery.
  JOURNAL   Nucleic Acids Res. 2025 Aug 11;53(15):gkaf809. doi: 
            10.1093/nar/gkaf809.
  PUBMED    40823809
REFERENCE   2  (bases 1 to 3426)
  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: "Nucleic 
            Acids Res."; date: "2025-08-11"; volume: "53(15):gkaf809. doi"; 
            pages: " 10.1093/nar/gkaf809"
COMMENT     Sequence Label: attB-GT-mNeonGreen-linker::pUC-GW-Kan_mod
FEATURES             Location/Qualifiers
     source          1..3426
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     657..676
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     promoter        complement(750..854)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     922..940
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(978..1000)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     1100..1119
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1313..1335
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1327..1344
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1328..1344
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1373..1409
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attB site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     primer_bind     complement(2199..2215)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2199..2215)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2212..2234)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2223..2239
                     /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(2247..2253,2254..2271,2272..2277))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2292..2313
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2430..2447)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2601..3189)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2681..2700)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3360..3426,1..749))
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(join(3360..3426,1..749))
                     /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="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
ORIGIN
        1 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
       61 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
      121 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
      181 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
      241 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
      301 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
      361 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
      421 aatacctgga atgctgtttt cccagggatc gcagtggtga gtaaccatgc atcatcagga
      481 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
      541 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
      601 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
      661 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
      721 caagacgttt cccgttgaat atggctcata ctcttccttt ttcaatatta ttgaagcatt
      781 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
      841 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
      901 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
      961 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     1021 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     1081 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg
     1141 tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc
     1201 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
     1261 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
     1321 cgacgttgta aaacgacggc cagtgaattg acgcgtattg ggatgaagac aagcttgtcg
     1381 acgacggcgg tctccgtcgt caggatcatc cggatggtga gcaagggcga ggaggataac
     1441 atggcctccc tgccagcaac acacgaactg cacatttttg gatcaattaa tggcgtggat
     1501 tttgatatgg tgggccaggg cacaggcaat cccaatgatg ggtatgaaga actgaacctg
     1561 aagagcacaa agggcgacct ccagttttct ccctggattc tggtgcccca tatcggatat
     1621 ggatttcacc agtacctccc ttatcctgat ggaatgagcc ctttccaggc agctatggtg
     1681 gatggctccg ggtatcaggt gcaccggaca atgcagtttg aagatggcgc ttctctgaca
     1741 gtgaattatc ggtatactta tgaaggatct catatcaaag gcgaggccca ggtgaaagga
     1801 acaggattcc ctgccgatgg gccagtgatg acaaattctc tgaccgcagc cgattggtgc
     1861 aggagcaaaa agacttaccc caacgataag actatcatct ctacttttaa atggtcatat
     1921 accactggca atgggaagag gtacaggtct actgctagga caacttacac atttgccaaa
     1981 cccatggcag ctaattatct gaaaaatcag cctatgtatg tgtttcgcaa gacagagctg
     2041 aaacatagca aaaccgagct gaactttaag gagtggcaga aagcctttac tgacgtgatg
     2101 gggatggatg aactttacaa gggaggctcc ggaggtggct ctggaaaggc cggcttgagt
     2161 cttcatccca atggcgcgcc gagcttggct cgagcatggt catagctgtt tcctgtgtga
     2221 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     2281 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     2341 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     2401 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2461 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2521 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2581 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2641 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2701 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     2761 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     2821 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     2881 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     2941 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3001 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     3061 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3121 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3181 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3241 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3301 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3361 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata
     3421 ccatat
//
