LOCUS       sequence-465759-        5347 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing the Insert 1 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 5347)
  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 5347)
  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: POC1426
FEATURES             Location/Qualifiers
     source          1..5347
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(244..255)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    1119..1143
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1299..2447)
                     /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(2783..3371)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2863..2882)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3542..4336)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     promoter        complement(4337..4441)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4528..5159
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5251..5275
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 cactctgtgg tgcagtacct ctcacgactt ggtctcccgt tgtggggctt agcttcatat
       61 tttcaaactg aaatattctc ttccttaacc tccacataaa tccaagttta taatttttat
      121 tattttaaaa ttttatttat ttttctgttt tggggacagg gtctcctcct gtcactcagg
      181 ctggagtgca atggcacaat catagctcac tgcagcctgg aactcctggg cttaagcaat
      241 cttcctgcct tcgattccca aagagctggg attatagtca tgaaccactg caatccaccc
      301 aaatccaagt ttacactaaa agataaaatt ccaacattgt aggggattgg tcaggtggtg
      361 ggaataatta taaagataaa gttataggaa atagacacaa accttcttgg aaggtggaaa
      421 gttttgcaaa agcctcagga tagggttata gctgaaagca gcctaatccc cttaccttga
      481 gttaatagct tcgagtaagt acaaagacat gtaagagagt ttatctaaag agcatgttta
      541 cctttgatca tttgtaggac tgctctctct gggggactgc gaccagatta attacccaca
      601 ggtgtgttga ctcaaagcct ttgtcattaa atctgtgctg aataaaggcc cacagggcca
      661 gatagtcagg gcacgcagct gccacaaccc tttctgtgag tggcctggcc ctctggtgca
      721 ctctttcact gaatatcggt gtctgagtac attattcatc catcgtgcag cctgggtctg
      781 ccggtcagac cctggcacaa catttaagag gaaatgaaag tcacaaagtt atcccagtct
      841 ctggagtcac tgtcaaaact ttggtgagga atcttccagg ttttccccta cttcaaatat
      901 atattaatat tatgtaagtg atattagtgg cattttcgcc caggctggaa tgcagtggca
      961 tgatctcggc tcactctaac ctctacctcc cagattcaag cgattctcct gcctcagcct
     1021 cccaaacagc tggaactaca ggcacccacc accacgcccg gcttgagacc aagtcgtgag
     1081 aggtactgca ccacgaagtg gtttaaacta tcagtgtttg acaggatata ttggcgggta
     1141 aacctaagag aaaagagcgt ttattagaat aatcggatat ttaaaagggc gtgaaaaggt
     1201 ttatccgttc gtccatttgt atgtgcatgc caaccacagg gttccccaga tcaggcgctg
     1261 gctgctgaac ccccagccgg aactgacccc acaaggccct agcgtttgca atgcaccagg
     1321 tcatcattga cccaggcgtg ttccaccagg ccgctgcctc gcaactcttc gcaggcttcg
     1381 ccgacctgct cgcgccactt cttcacgcgg gtggaatccg atccgcacat gaggcggaag
     1441 gtttccagct tgagcgggta cggctcccgg tgcgagctga aatagtcgaa catccgtcgg
     1501 gccgtcggcg acagcttgcg gtacttctcc catatgaatt tcgtgtagtg gtcgccagca
     1561 aacagcacga cgatttcctc gtcgatcagg acctggcaac gggacgtttt cttgccacgg
     1621 tccaggacgc ggaagcggtg cagcagcgac accgattcca ggtgcccaac gcggtcggac
     1681 gtgaagccca tcgccgtcgc ctgtaggcgc gacaggcatt cctcggcctt cgtgtaatac
     1741 cggccattga tcgaccagcc caggtcctgg caaagctcgt agaacgtgaa ggtgatcggc
     1801 tcgccgatag gggtgcgctt cgcgtactcc aacacctgct gccacaccag ttcgtcatcg
     1861 tcggcccgca gctcgacgcc ggtgtaggtg atcttcacgt ccttgttgac gtggaaaatg
     1921 accttgtttt gcagcgcctc gcgcgggatt ttcttgttgc gcgtggtgaa cagggcagag
     1981 cgggccgtgt cgtttggcat cgctcgcatc gtgtccggcc acggcgcaat atcgaacaag
     2041 gaaagctgca tttccttgat ctgctgcttc gtgtgtttca gcaacgcggc ctgcttggcc
     2101 tcgctgacct gttttgccag gtcctcgccg gcggtttttc gcttcttggt cgtcatagtt
     2161 cctcgcgtgt cgatggtcat cgacttcgcc aaacctgccg cctcctgttc aagacgacgc
     2221 gaacgctcca cggcggccga tggcgcgggc agggcagggg gagccagttg cacgctgtcg
     2281 cgctcgatct tggccgtagc ttgctggacc atcgagccga cggactggaa ggtttcgcgg
     2341 ggcgcacgca tgacggtgcg gcttgcgatg gtttcggcat cctcggcgga aaaccccgcg
     2401 tcgatcagtt cttgcctgta tgccttccgg tcaaacgtcc gattcattca ccctccttgc
     2461 gggattgccc cgactcacgc cggggcaatg tgcccttatt cctgatttga cccgcctggt
     2521 gccttggtgt ccagataatc caccttatcg gcaatgaagt cggtcccgta gaccgtctgg
     2581 ccgtccttct cgtacttggt attccgaatc ttgccctgca cgaataccag cgaccccttg
     2641 cccaaatact tgccgtgggc ctcggcctga gagccaaaac acttgatgcg gaagaagtcg
     2701 gtgcgctcct gcttgtcgcc ggcatcgttg cgccacatct aggatctgcc aggaaccgta
     2761 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     2821 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     2881 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     2941 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3001 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3061 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3121 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3181 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3241 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3301 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3361 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3421 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3481 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     3541 gctaaaacaa ttcatccagt aaaatataat attttatttt ctcccaatca ggcttgatcc
     3601 ccagtaagtc aaaaaatagc tcgacatact gttcttcccc gatatcctcc ctgagcgacc
     3661 ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc gcttctccca agatcaataa
     3721 agccacttac tttgccatct ttcacaaaga tgttgctgtc tcccaggtcg ccgtgggaaa
     3781 agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc atacagctcg cgcggatctt
     3841 taaatggagt gtcctcttcc cagttttcgc aatccacatc ggccagatcg ttattcagta
     3901 agtaatccaa ttcggctaag cggctgtcta agctattcgt atagggacaa tccgatatgt
     3961 cgatggagtg aaagagcctg atgcactccg catacagctc gataatcttt tcagggcttt
     4021 gttcatcttc atactcttcc gagcaaagga cgccatcggc ctcactcatg agcagattgc
     4081 tccagccatc atgccgttca aagtgcagga cctttggaac aggcagcttt ccttccagcc
     4141 atagcatcat gtccttttcc cgttccacat cataggtggt ccctttatac cggctgtccg
     4201 tcatttttaa atataggttt tcattttctc ccaccagctt atatacctta gcaggagaca
     4261 ttccttccgt atcttttacg cagcggtatt tttcgatcag ttttttcaat tccggtgata
     4321 ttctcatttt agccatactc ttcctttttc aatattattg aagcatttat cagggttatt
     4381 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     4441 gcacgaattg gccagcgctg ccatttttgg ggtgaggccg ttcgcggccg aggggcgcag
     4501 cccctggggg gatgggaggc ccgcgttagc gggccgggag ggttcgagaa gggggggcac
     4561 cccccttcgg cgtgcgcggt cacgcgcaca gggcgcagcc ctggttaaaa acaaggttta
     4621 taaatattgg tttaaaagca ggttaaaaga caggttagcg gtggccgaaa aacgggcgga
     4681 aacccttgca aatgctggat tttctgcctg tggacagccc ctcaaatgtc aataggtgcg
     4741 cccctcatct gtcagcactc tgcccctcaa gtgtcaagga tcgcgcccct catctgtcag
     4801 tagtcgcgcc cctcaagtgt caataccgca gggcacttat ccccaggctt gtccacatca
     4861 tctgtgggaa actcgcgtaa aatcaggcgt tttcgccgat ttgcgaggct ggccagctcc
     4921 acgtcgccgg ccgaaatcga gcctgcccct catctgtcaa cgccgcgccg ggtgagtcgg
     4981 cccctcaagt gtcaacgtcc gcccctcatc tgtcagtgag ggccaagttt tccgcgaggt
     5041 atccacaacg ccggcggccg cggtgtctcg cacacggctt cgacggcgtt tctggcgcgt
     5101 ttgcagggcc atagacggcc gccagcccag cggcgagggc aaccagcccg gtgagcgtcg
     5161 caaaggagat cctgatctga ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag
     5221 ctgccggtcg gggagctgtt ggctggctgg tggcaggata tattgtggtg taaacaaatt
     5281 gacgcttaga caacttaata acacattgcg gacgttttta atgtactggg gtggatgcag
     5341 tgggccc
//
