LOCUS       sequence-465760-        5214 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing the Insert 4 to be assembled in POC1430 using the
            site-selective methylation protection approach.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5214)
  AUTHORS   Flores-Fernandez CN, Lin D, Robins K, O'Callaghan CA
  TITLE     UniClo: scarless hierarchical DNA assembly without sequence 
            constraint.
  JOURNAL   Nucleic Acids Res. 2025 Jun 20;53(12):gkaf548. doi: 
            10.1093/nar/gkaf548.
  PUBMED    40548934
REFERENCE   2  (bases 1 to 5214)
  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: "2025-06-20"; volume: "53(12):gkaf548. doi"; 
            pages: " 10.1093/nar/gkaf548"
COMMENT     Sequence Label: POC1429
FEATURES             Location/Qualifiers
     source          1..5214
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    986..1010
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1166..2314)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
                     MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      complement(2650..3238)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2730..2749)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3409..4203)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     promoter        complement(4204..4308)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4395..5026
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5118..5142
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 cactctgtgg tgcagtacct ctcacgactt ggtctcatag accagggcgg ggtgacgggt
       61 gatcccagtc ctcgcagtga attccgggca gcaaaattca aaacacatgc ggccaaggcc
      121 gggcacggtg gttcacgcct gtaatcccag cactttggga ggtcgaggcg ggcgatcacc
      181 tgaggtcggg agctcgagac caacctgacc aacatgggga aatcccgtct ctactaaaaa
      241 tataaaatta gacgggcttg gtggtgaatg cctgtaatcc cagctagtcg ggaggctgag
      301 gcaggagaat cgcttaaacc ttggaggcgg aggttgcggt gagccgagat cgcgccattg
      361 cacttcagcc tgggcaacaa gagggaaaac tccgtcgcaa aaactttcgg gggcggagcg
      421 gagccccgcc ctgggttatg taagcgaccg cgctgggccg tttctctttc ttttccggac
      481 cctgcagtgg cgcctaaagt ctgagagagg gaagtcgcct ctgtgctcgt gagtgcatgg
      541 ggtataaggc aagtgctgag ggagaaaacg tagttgatgg ggtagagcag acggggttgg
      601 aggtggggtg gagggggagg gctttggaca gaagacctgg gaggcttggt gggggagggg
      661 cgcccaggcc tgggcactaa gaaacaagtc ccctggagct caagaccatc tcggcctccc
      721 ctagcccaag agaggactgg cttcatgact ccctgaaacc atttctaaat gccttagaac
      781 aaaccttgca tattcattat tgttattgaa ctattaaaag tcttttttgg gggcgagctg
      841 aatcagatcc tttgctggag ctggcacacg gaggaagtcc tggagggagg gtagacaccg
      901 tggaggtaag ggcttgggac ctgtgtcagg tgagaccaag tcgtgagagg tactgcacca
      961 cgaagtggtt taaactatca gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa
     1021 agagcgttta ttagaataat cggatattta aaagggcgtg aaaaggttta tccgttcgtc
     1081 catttgtatg tgcatgccaa ccacagggtt ccccagatca ggcgctggct gctgaacccc
     1141 cagccggaac tgaccccaca aggccctagc gtttgcaatg caccaggtca tcattgaccc
     1201 aggcgtgttc caccaggccg ctgcctcgca actcttcgca ggcttcgccg acctgctcgc
     1261 gccacttctt cacgcgggtg gaatccgatc cgcacatgag gcggaaggtt tccagcttga
     1321 gcgggtacgg ctcccggtgc gagctgaaat agtcgaacat ccgtcgggcc gtcggcgaca
     1381 gcttgcggta cttctcccat atgaatttcg tgtagtggtc gccagcaaac agcacgacga
     1441 tttcctcgtc gatcaggacc tggcaacggg acgttttctt gccacggtcc aggacgcgga
     1501 agcggtgcag cagcgacacc gattccaggt gcccaacgcg gtcggacgtg aagcccatcg
     1561 ccgtcgcctg taggcgcgac aggcattcct cggccttcgt gtaataccgg ccattgatcg
     1621 accagcccag gtcctggcaa agctcgtaga acgtgaaggt gatcggctcg ccgatagggg
     1681 tgcgcttcgc gtactccaac acctgctgcc acaccagttc gtcatcgtcg gcccgcagct
     1741 cgacgccggt gtaggtgatc ttcacgtcct tgttgacgtg gaaaatgacc ttgttttgca
     1801 gcgcctcgcg cgggattttc ttgttgcgcg tggtgaacag ggcagagcgg gccgtgtcgt
     1861 ttggcatcgc tcgcatcgtg tccggccacg gcgcaatatc gaacaaggaa agctgcattt
     1921 ccttgatctg ctgcttcgtg tgtttcagca acgcggcctg cttggcctcg ctgacctgtt
     1981 ttgccaggtc ctcgccggcg gtttttcgct tcttggtcgt catagttcct cgcgtgtcga
     2041 tggtcatcga cttcgccaaa cctgccgcct cctgttcaag acgacgcgaa cgctccacgg
     2101 cggccgatgg cgcgggcagg gcagggggag ccagttgcac gctgtcgcgc tcgatcttgg
     2161 ccgtagcttg ctggaccatc gagccgacgg actggaaggt ttcgcggggc gcacgcatga
     2221 cggtgcggct tgcgatggtt tcggcatcct cggcggaaaa ccccgcgtcg atcagttctt
     2281 gcctgtatgc cttccggtca aacgtccgat tcattcaccc tccttgcggg attgccccga
     2341 ctcacgccgg ggcaatgtgc ccttattcct gatttgaccc gcctggtgcc ttggtgtcca
     2401 gataatccac cttatcggca atgaagtcgg tcccgtagac cgtctggccg tccttctcgt
     2461 acttggtatt ccgaatcttg ccctgcacga ataccagcga ccccttgccc aaatacttgc
     2521 cgtgggcctc ggcctgagag ccaaaacact tgatgcggaa gaagtcggtg cgctcctgct
     2581 tgtcgccggc atcgttgcgc cacatctagg atctgccagg aaccgtaaaa aggccgcgtt
     2641 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     2701 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     2761 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     2821 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     2881 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     2941 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     3001 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     3061 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     3121 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     3181 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     3241 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     3301 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     3361 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagct aaaacaattc
     3421 atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa
     3481 aaatagctcg acatactgtt cttccccgat atcctccctg agcgaccgga cgcagaaggc
     3541 aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc cacttacttt
     3601 gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga caagttcctc
     3661 ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa atggagtgtc
     3721 ctcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt aatccaattc
     3781 ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga tggagtgaaa
     3841 gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt catcttcata
     3901 ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc agccatcatg
     3961 ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata gcatcatgtc
     4021 cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca tttttaaata
     4081 taggttttca ttttctccca ccagcttata taccttagca ggagacattc cttccgtatc
     4141 ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc tcattttagc
     4201 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4261 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca cgaattggcc
     4321 agcgctgcca tttttggggt gaggccgttc gcggccgagg ggcgcagccc ctggggggat
     4381 gggaggcccg cgttagcggg ccgggagggt tcgagaaggg ggggcacccc ccttcggcgt
     4441 gcgcggtcac gcgcacaggg cgcagccctg gttaaaaaca aggtttataa atattggttt
     4501 aaaagcaggt taaaagacag gttagcggtg gccgaaaaac gggcggaaac ccttgcaaat
     4561 gctggatttt ctgcctgtgg acagcccctc aaatgtcaat aggtgcgccc ctcatctgtc
     4621 agcactctgc ccctcaagtg tcaaggatcg cgcccctcat ctgtcagtag tcgcgcccct
     4681 caagtgtcaa taccgcaggg cacttatccc caggcttgtc cacatcatct gtgggaaact
     4741 cgcgtaaaat caggcgtttt cgccgatttg cgaggctggc cagctccacg tcgccggccg
     4801 aaatcgagcc tgcccctcat ctgtcaacgc cgcgccgggt gagtcggccc ctcaagtgtc
     4861 aacgtccgcc cctcatctgt cagtgagggc caagttttcc gcgaggtatc cacaacgccg
     4921 gcggccgcgg tgtctcgcac acggcttcga cggcgtttct ggcgcgtttg cagggccata
     4981 gacggccgcc agcccagcgg cgagggcaac cagcccggtg agcgtcgcaa aggagatcct
     5041 gatctgactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg
     5101 agctgttggc tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa
     5161 cttaataaca cattgcggac gtttttaatg tactggggtg gatgcagtgg gccc
//
