LOCUS       sequence-69074-e        5888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5888)
  AUTHORS   Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S
  TITLE     A modular cloning system for standardized assembly of multigene 
            constructs.
  JOURNAL   PLoS One. 2011 Feb 18;6(2):e16765. doi: 
            10.1371/journal.pone.0016765.
  PUBMED    21364738
REFERENCE   2  (bases 1 to 5888)
  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: "PLoS One.";
            date: "2011-02-18"; volume: "6(2):e16765. doi"; pages: " 
            10.1371/journal.pone.0016765"
COMMENT     Sequence Label: pICH82094
FEATURES             Location/Qualifiers
     source          1..5888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    26..50
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             436..1584
                     /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      1635..2266
                     /label=oriV
                     /note="incP origin of replication"
     promoter        2351..2455
                     /gene="bla"
                     /label=AmpR promoter
     CDS             2456..3250
                     /codon_start=1
                     /gene="aphA-3"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin"
                     /translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
                     TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
                     EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
                     PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
                     FCVRSLREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
     rep_origin      3272..3817
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     misc_feature    4191..4215
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     rep_origin      4473..5061
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    5349..5370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5385..5390,5391..5408,5409..5415)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5423..5439
                     /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)."
     primer_bind     5428..5450
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5447..5463
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5447..5463
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5459..5782
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    5476..5532
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(5533..5550)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5533..5549)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5542..5564)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5758..5777)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtgatccgtt taaactatca gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa
       61 agagcgttta ttagaataat cggatattta aaagggcgtg aaaaggttta tccgttcgtc
      121 catttgtatg tgccagccgc ctagatgtgg cgcaacgatg ccggcgacaa gcaggagcgc
      181 accgacttct tccgcatcaa gtgttttggc tctcaggccg aggcccacgg caagtatttg
      241 ggcaaggggt cgctggtatt cgtgcagggc aagattcgga ataccaagta cgagaaggac
      301 ggccagacgg tctacgggac cgacttcatt gccgataagg tggattatct ggacaccaag
      361 gcaccaggcg ggtcaaatca ggaataaggg cacattgccc cggcgtgagt cggggcaatc
      421 ccgcaaggag ggtgaatgaa tcggacgttt gaccggaagg catacaggca agaactgatc
      481 gacgcggggt tttccgccga ggatgccgaa accatcgcaa gccgcaccgt catgcgtgcg
      541 ccccgcgaaa ccttccagtc cgtcggctcg atggtccagc aagctacggc caagatcgag
      601 cgcgacagcg tgcaactggc tccccctgcc ctgcccgcgc catcggccgc cgtggagcgt
      661 tcgcgtcgtc ttgaacagga ggcggcaggt ttggcgaagt cgatgaccat cgacacgcga
      721 ggaactatga cgaccaagaa gcgaaaaacc gccggcgagg acctggcaaa acaggtcagc
      781 gaggccaagc aggccgcgtt gctgaaacac acgaagcagc agatcaagga aatgcagctt
      841 tccttgttcg atattgcgcc gtggccggac acgatgcgag cgatgccaaa cgacacggcc
      901 cgctctgccc tgttcaccac gcgcaacaag aaaatcccgc gcgaggcgct gcaaaacaag
      961 gtcattttcc acgtcaacaa ggacgtgaag atcacctaca ccggcgtcga gctgcgggcc
     1021 gacgatgacg aactggtgtg gcagcaggtg ttggagtacg cgaagcgcac ccctatcggc
     1081 gagccgatca ccttcacgtt ctacgagctt tgccaggacc tgggctggtc gatcaatggc
     1141 cggtattaca cgaaggccga ggaatgcctg tcgcgcctac aggcgacggc gatgggcttc
     1201 acgtccgacc gcgttgggca cctggaatcg gtgtcgctgc tgcaccgctt ccgcgtcctg
     1261 gaccgtggca agaaaacgtc ccgttgccag gtcctgatcg acgaggaaat cgtcgtgctg
     1321 tttgctggcg accactacac gaaattcata tgggagaagt accgcaagct gtcgccgacg
     1381 gcccgacgga tgttcgacta tttcagctcg caccgggagc cgtacccgct caagctggaa
     1441 accttccgcc tcatgtgcgg atcggattcc acccgcgtga agaagtggcg cgagcaggtc
     1501 ggcgaagcct gcgaagagtt gcgaggcagc ggcctggtgg aacacgcctg ggtcaatgat
     1561 gacctggtgc attgcaaacg ctagggcctt gtggggtcag ttccggctgg gggttcagca
     1621 gcccaaccct ttgcgacgct caccgggctg gttgccctcg ccgctgggct ggcggccgtc
     1681 tatggccctg caaacgcgcc agaaacgccg tcgaagccgt gtgcgagaca ccgcggccgc
     1741 cggcgttgtg gatacctcgc ggaaaacttg gccctcactg acagatgagg ggcggacgtt
     1801 gacacttgag gggccgactc acccggcgcg gcgttgacag atgaggggca ggctcgattt
     1861 cggccggcga cgtggagctg gccagcctcg caaatcggcg aaaacgcctg attttacgcg
     1921 agtttcccac agatgatgtg gacaagcctg gggataagtg ccctgcggta ttgacacttg
     1981 aggggcgcga ctactgacag atgaggggcg cgatccttga cacttgaggg gcagagtgct
     2041 gacagatgag gggcgcacct attgacattt gaggggctgt ccacaggcag aaaatccagc
     2101 atttgcaagg gtttccgccc gtttttcggc caccgctaac ctgtctttta acctgctttt
     2161 aaaccaatat ttataaacct tgtttttaac cagggctgcg ccctgtgcgc gtgaccgcgc
     2221 acgccgaagg ggggtgcccc cccttctcga accctcccgg cccgctaacg cgggcctccc
     2281 atccccccag gggctgcgcc cctcggccgc gaacggcctc accccaaaaa tggcagcgct
     2341 ggcccgagtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     2401 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatggc
     2461 taaaatgaga atatcaccgg aattgaaaaa actgatcgaa aaataccgct gcgtaaaaga
     2521 tacggaagga atgtctcctg ctaaggtata taagctggtg ggagaaaatg aaaacctata
     2581 tttaaaaatg acggacagcc ggtataaagg gaccacctat gatgtggaac gggaaaagga
     2641 catgatgcta tggctggaag gaaagctgcc tgttccaaag gtcctgcact ttgaacggca
     2701 tgatggctgg agcaatctgc tcatgagtga ggccgatggc gtcctttgct cggaagagta
     2761 tgaagatgaa caaagccctg aaaagattat cgagctgtat gcggagtgca tcaggctctt
     2821 tcactccatc gacatatcgg attgtcccta tacgaatagc ttagacagcc gcttagccga
     2881 attggattac ttactgaata acgatctggc cgatgtggat tgcgaaaact gggaagagga
     2941 cactccattt aaagatccgc gcgagctgta tgatttttta aagacggaaa agcccgaaga
     3001 ggaacttgtc ttttcccacg gcgacctggg agacagcaac atctttgtga aagatggcaa
     3061 agtaagtggc tttattgatc ttgggagaag cggcagggcg gacaagtggt atgacattgc
     3121 cttctgcgtc cggtcgctca gggaggatat cggggaagaa cagtatgtcg agctattttt
     3181 tgacttactg gggatcaagc ctgattggga gaaaataaaa tattatattt tactggatga
     3241 attgttttag ctgtttaata agatgatctt cttgagatcg ttttggtctg cgcgtaatct
     3301 cttgctctga aaacgaaaaa accgccttgc agggcggttt ttcgaaggtt ctctgagcta
     3361 ccaactcttt gaaccgaggt aactggcttg gaggagcgca gtcaccaaaa cttgtccttt
     3421 cagtttagcc ttaaccggcg catgacttca agactaactc ctctaaatca attaccagtg
     3481 gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg actcaagacg atagttaccg
     3541 gataaggcgc agcggtcgga ctgaacgggg ggttcgtgca tacagtccag cttggagcga
     3601 actgcctacc cggaactgag tgtcaggcgt ggaatgagac aaacgcggcc ataacagcgg
     3661 aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc acgagggagc cgccaggggg
     3721 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac cactgatttg agcgtcagat
     3781 ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaac ggctttgccg cggccctctc
     3841 acttccctgt taagtatctt cctggcatct tccaggaaat ctccgccccg ttcgtaagcc
     3901 atttccgctc gccgcagtcg aacgaccgag cgtagcgagt cagtgagcga ggaagcggaa
     3961 tatatcctgt atcacatatt ctgctgacgc accggtgcag ccttttttct cctgccacat
     4021 gaagcacttc actgacaccc tcatcagtgc caacatagta agccagtata cactccgcta
     4081 gcgccctgct cggatctgtt ggaccggaca gtagtcatgg ttgatgggct gcctgtatcg
     4141 agtggtgatt ttgtgccgag ctgccggtcg gggagctgtt ggctggctgg tggcaggata
     4201 tattgtggtg taaacaaatt gacgcttaga caacttaata acacattgcg gacgttttta
     4261 atgtactggg gttgaacact ctgtgaagac aatgcctgag acctgtcaga ccaagtttac
     4321 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
     4381 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
     4441 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc
     4501 tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag
     4561 ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc
     4621 cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac
     4681 ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc
     4741 gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt
     4801 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt
     4861 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc
     4921 ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt
     4981 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
     5041 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
     5101 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
     5161 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag
     5221 tcagtgagcg aggaagcgga tgagcgccca atacgcaaac cgcctctccc cgcgcgttgg
     5281 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     5341 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     5401 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     5461 gaccatgatt acgccaagct tgcatgcctg caggtcgact ctagaggatc cccgggtacc
     5521 gagctcgaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     5581 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     5641 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg
     5701 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac
     5761 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc
     5821 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga cggtctcagg
     5881 gacacgaa
//
