LOCUS       sequence-465764-        5175 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid containing the Insert 3 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 5175)
  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 5175)
  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: POC1428
FEATURES             Location/Qualifiers
     source          1..5175
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    947..971
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(1127..2275)
                     /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(2611..3199)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2691..2710)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3370..4164)
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     promoter        complement(4165..4269)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4356..4987
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    5079..5103
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 cactctgtgg tgcagtacct ctcacgactt ggtctcactc cagaggctga ggcagcagaa
       61 tgccctgaac ccgggaggcg gagcttgcag tgagccaaga tcgcgccact gcactccagc
      121 ctgggcgaca gagcaagact ccgtctcaaa aaaaaaaaac aaaaaacaaa aaaactctcc
      181 tttacttttt ctctcccctt ttcttcctat ctcttccctc atttcttcaa cacgtccccc
      241 catccttccc tcttttctcc attctctgca tttgatcccc ggtatattcc agcctccagg
      301 ccaacaaact tctccgcgtc cgccgggagc aggtcaggga agggacgcga ggcggcgctg
      361 tcaccgcatt ctgagcgccg cagctccctg ggccccttgt atcatttcag tgaaggtcac
      421 tccagtcttt catggaggcc aaactaaggg tgtaaattag gatcctcact gaagtggcgg
      481 gaccctaaga ggctttttcc tggcccctta gttgtgggtt ttcctgcggg cggcgcagcc
      541 ggtttccatc agaaccgccc agaggcggac gctgccttcc tggggtgacg gagcagcagg
      601 aagcgttttc ggatcctgga atacgtgggc ggcccgtggg aggggctgag gcgcagtttc
      661 ctactcaccc ggatccgaat cctccgcggt gctgtttcaa gagagccgga ttccagatcg
      721 cgctccagcc cggactcgga attcctgccc tgcgggtctg cattttcata acgggcaggt
      781 gtgagtgccc tgcagctgga gaccagaagc ctgaaggcag ctcggccctc cccagcccac
      841 agcgccgtta ttccgtttct atatcagtaa acacatttca ttttccgtag atgagaccaa
      901 gtcgtgagag gtactgcacc acgaagtggt ttaaactatc agtgtttgac aggatatatt
      961 ggcgggtaaa cctaagagaa aagagcgttt attagaataa tcggatattt aaaagggcgt
     1021 gaaaaggttt atccgttcgt ccatttgtat gtgcatgcca accacagggt tccccagatc
     1081 aggcgctggc tgctgaaccc ccagccggaa ctgaccccac aaggccctag cgtttgcaat
     1141 gcaccaggtc atcattgacc caggcgtgtt ccaccaggcc gctgcctcgc aactcttcgc
     1201 aggcttcgcc gacctgctcg cgccacttct tcacgcgggt ggaatccgat ccgcacatga
     1261 ggcggaaggt ttccagcttg agcgggtacg gctcccggtg cgagctgaaa tagtcgaaca
     1321 tccgtcgggc cgtcggcgac agcttgcggt acttctccca tatgaatttc gtgtagtggt
     1381 cgccagcaaa cagcacgacg atttcctcgt cgatcaggac ctggcaacgg gacgttttct
     1441 tgccacggtc caggacgcgg aagcggtgca gcagcgacac cgattccagg tgcccaacgc
     1501 ggtcggacgt gaagcccatc gccgtcgcct gtaggcgcga caggcattcc tcggccttcg
     1561 tgtaataccg gccattgatc gaccagccca ggtcctggca aagctcgtag aacgtgaagg
     1621 tgatcggctc gccgataggg gtgcgcttcg cgtactccaa cacctgctgc cacaccagtt
     1681 cgtcatcgtc ggcccgcagc tcgacgccgg tgtaggtgat cttcacgtcc ttgttgacgt
     1741 ggaaaatgac cttgttttgc agcgcctcgc gcgggatttt cttgttgcgc gtggtgaaca
     1801 gggcagagcg ggccgtgtcg tttggcatcg ctcgcatcgt gtccggccac ggcgcaatat
     1861 cgaacaagga aagctgcatt tccttgatct gctgcttcgt gtgtttcagc aacgcggcct
     1921 gcttggcctc gctgacctgt tttgccaggt cctcgccggc ggtttttcgc ttcttggtcg
     1981 tcatagttcc tcgcgtgtcg atggtcatcg acttcgccaa acctgccgcc tcctgttcaa
     2041 gacgacgcga acgctccacg gcggccgatg gcgcgggcag ggcaggggga gccagttgca
     2101 cgctgtcgcg ctcgatcttg gccgtagctt gctggaccat cgagccgacg gactggaagg
     2161 tttcgcgggg cgcacgcatg acggtgcggc ttgcgatggt ttcggcatcc tcggcggaaa
     2221 accccgcgtc gatcagttct tgcctgtatg ccttccggtc aaacgtccga ttcattcacc
     2281 ctccttgcgg gattgccccg actcacgccg gggcaatgtg cccttattcc tgatttgacc
     2341 cgcctggtgc cttggtgtcc agataatcca ccttatcggc aatgaagtcg gtcccgtaga
     2401 ccgtctggcc gtccttctcg tacttggtat tccgaatctt gccctgcacg aataccagcg
     2461 accccttgcc caaatacttg ccgtgggcct cggcctgaga gccaaaacac ttgatgcgga
     2521 agaagtcggt gcgctcctgc ttgtcgccgg catcgttgcg ccacatctag gatctgccag
     2581 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     2641 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     2701 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     2761 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     2821 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     2881 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     2941 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     3001 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     3061 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     3121 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     3181 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     3241 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     3301 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     3361 gtctgacagc taaaacaatt catccagtaa aatataatat tttattttct cccaatcagg
     3421 cttgatcccc agtaagtcaa aaaatagctc gacatactgt tcttccccga tatcctccct
     3481 gagcgaccgg acgcagaagg caatgtcata ccacttgtcc gccctgccgc ttctcccaag
     3541 atcaataaag ccacttactt tgccatcttt cacaaagatg ttgctgtctc ccaggtcgcc
     3601 gtgggaaaag acaagttcct cttcgggctt ttccgtcttt aaaaaatcat acagctcgcg
     3661 cggatcttta aatggagtgt cctcttccca gttttcgcaa tccacatcgg ccagatcgtt
     3721 attcagtaag taatccaatt cggctaagcg gctgtctaag ctattcgtat agggacaatc
     3781 cgatatgtcg atggagtgaa agagcctgat gcactccgca tacagctcga taatcttttc
     3841 agggctttgt tcatcttcat actcttccga gcaaaggacg ccatcggcct cactcatgag
     3901 cagattgctc cagccatcat gccgttcaaa gtgcaggacc tttggaacag gcagctttcc
     3961 ttccagccat agcatcatgt ccttttcccg ttccacatca taggtggtcc ctttataccg
     4021 gctgtccgtc atttttaaat ataggttttc attttctccc accagcttat ataccttagc
     4081 aggagacatt ccttccgtat cttttacgca gcggtatttt tcgatcagtt ttttcaattc
     4141 cggtgatatt ctcattttag ccatactctt cctttttcaa tattattgaa gcatttatca
     4201 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     4261 ggttccgcgc acgaattggc cagcgctgcc atttttgggg tgaggccgtt cgcggccgag
     4321 gggcgcagcc cctgggggga tgggaggccc gcgttagcgg gccgggaggg ttcgagaagg
     4381 gggggcaccc cccttcggcg tgcgcggtca cgcgcacagg gcgcagccct ggttaaaaac
     4441 aaggtttata aatattggtt taaaagcagg ttaaaagaca ggttagcggt ggccgaaaaa
     4501 cgggcggaaa cccttgcaaa tgctggattt tctgcctgtg gacagcccct caaatgtcaa
     4561 taggtgcgcc cctcatctgt cagcactctg cccctcaagt gtcaaggatc gcgcccctca
     4621 tctgtcagta gtcgcgcccc tcaagtgtca ataccgcagg gcacttatcc ccaggcttgt
     4681 ccacatcatc tgtgggaaac tcgcgtaaaa tcaggcgttt tcgccgattt gcgaggctgg
     4741 ccagctccac gtcgccggcc gaaatcgagc ctgcccctca tctgtcaacg ccgcgccggg
     4801 tgagtcggcc cctcaagtgt caacgtccgc ccctcatctg tcagtgaggg ccaagttttc
     4861 cgcgaggtat ccacaacgcc ggcggccgcg gtgtctcgca cacggcttcg acggcgtttc
     4921 tggcgcgttt gcagggccat agacggccgc cagcccagcg gcgagggcaa ccagcccggt
     4981 gagcgtcgca aaggagatcc tgatctgact gatgggctgc ctgtatcgag tggtgatttt
     5041 gtgccgagct gccggtcggg gagctgttgg ctggctggtg gcaggatata ttgtggtgta
     5101 aacaaattga cgcttagaca acttaataac acattgcgga cgtttttaat gtactggggt
     5161 ggatgcagtg ggccc
//
