LOCUS       sequence-383215-        3867 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid is designed for use in a procedure to synthesize 
            single-stranded DNA containing only the insert..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3867)
  AUTHORS   Strawn IK, Steiner PJ, Newton MS, Baumer ZT, Whitehead TA
  TITLE     A method for generating user-defined circular single-stranded DNA 
            from plasmid DNA using Golden Gate intramolecular ligation.
  JOURNAL   Biotechnol Bioeng. 2023 Oct;120(10):3057-3066. doi: 
            10.1002/bit.28471. Epub 2023 Jun 27.
  PUBMED    37366288
REFERENCE   2  (bases 1 to 3867)
  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: "10.1002/bit.28471";
            journalName: "Biotechnol Bioeng"; date: "2023-10"; volume: "120"; 
            issue: "10"; pages: "3057-3066"
COMMENT     Sequence Label: pIS003
FEATURES             Location/Qualifiers
     source          1..3867
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(82..897)
                     /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(82..897)
                     /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"
     primer_bind     805..824
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     889..908
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1122..1710
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1611..1630
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    complement(1923..1956)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     protein_bind    complement(1923..1956)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1971..2004)
                     /label=lox5171
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    2534..2565
                     /label=rox
                     /bound_moiety="Dre recombinase"
     polyA_signal    2612..2733
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2623..2642)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2677..2696
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(join(2863..3576,3577..3579,3580..3582))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(2866..3576,3577..3579,3580..3582))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2908..2929)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3257..3276
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3516..3537
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    3664..3695
                     /label=rox
                     /bound_moiety="Dre recombinase"
     protein_bind    complement(3696..3729)
                     /label=loxN
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AGGTATAC) (Shaw et al., 2021). loxN sites are 
                     compatible with each other, but incompatible with loxP or 
                     lox2272 sites."
     protein_bind    complement(3696..3729)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(3776..3809)
                     /label=lox5171
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
ORIGIN
        1 catatcctct gccttgaggt cagacggagt gacaaatgag cgctgtgaga ccgcgtgtta
       61 caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt
      121 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
      181 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
      241 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
      301 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagcttat gcatttcttt
      361 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
      421 accgttattc attcgtgatt gcgcctgagc gaggcgaaat acgcgatcgc tgttaaaagg
      481 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
      541 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
      601 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
      661 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
      721 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
      781 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
      841 catgttggaa tttaatcgcg gcctggagca agacgtttcc cgttgaatat ggctcataac
      901 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt
      961 atcttgtgca atgtaacatc agagattttg agacacaacg tggctttgtt gaataaatcg
     1021 aacttttgct gagttgaagg atcagtcatg accaaaatcc cttaacgtga gttttcgttc
     1081 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     1141 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     1201 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     1261 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     1321 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     1381 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     1441 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     1501 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     1561 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     1621 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     1681 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     1741 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     1801 gataaccgtg gtctctgctg aggaggggaa gtgagtctca agattgcagt ggtagaagtc
     1861 aagatgtgat tgggaaagac tcaagttctt tctgccctct actcagccct gccaacccca
     1921 ggataacttc gtataggata ccttatacga agttatctca gctagaagcc ataacttcgt
     1981 atagtacaca ttatacgaag ttataagctt gacccaatcc ggtagattga agacctgagg
     2041 tgcatgcctc tgtcccctcc aaccccactc ctgcccctcc tgtgtctctc cctcctgccc
     2101 tacagtcggc cctgaacgag tagctatcct tcaagactct accacccgca cgggctgcac
     2161 tattaaagtg gacttcaaca tgtccctcac cctgtggtgt gtggcgcggt cttgccctga
     2221 gccagagtac gtgtgggcct tcaatggaaa ggccttgaag aatggccagg atcacctgaa
     2281 catcagcagt atgactgcag cccatgaggg cacgtacacg tgcatcgcta agaactccaa
     2341 gacgctgctg tctggctctg cctcagtggt agtcaagctg tccggtgagt cagccccagc
     2401 ctgatagcca cctgctctct agagaggccc agctggcctt tgctgactgg gctgccattt
     2461 tccagaaaat aattggattc ccagcacact cattggccag ttgtgacacg gtggcgtatc
     2521 agattgccct gtctaacttt aaataatgcc aattatttaa agttagttta tcgaattcct
     2581 gcagcccggt agatctttac aatttacgcg ttaagataca ttgatgagtt tggacaaacc
     2641 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta
     2701 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg
     2761 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt
     2821 ggtatggctg attatgatca gttatctaga tccggtggat ccttatttgt acagctcgtc
     2881 catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt gatcgcgctt
     2941 ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt cgggcagcag
     3001 cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggcgagct gcacgctgcc
     3061 gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct gcttgtcggc
     3121 catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc
     3181 gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt cgccctcgaa
     3241 cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg
     3301 gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta
     3361 gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc agggcacggg
     3421 cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc
     3481 gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct cgaccaggat
     3541 gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atccctggaa aaaagaaatg
     3601 cgaagattgc gcctgcgcat cgataaaata aaagatttta tttagtctcc agaaaaagcc
     3661 cggtaacttt aaataatgcc aattatttaa agttaataac ttcgtatagg ataccttata
     3721 cgaagttatc cattcaggct gtgctagcat caatggcatg gcacaaagct tagccataac
     3781 ttcgtatagt acacattata cgaagttatt tgtctaggtg cggaaagagg caggcaaagc
     3841 ctattgcata ggaaagaaat ggtgact
//
