LOCUS       sequence-69025-e        4945 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 4945)
  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 4945)
  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: pICH49277
FEATURES             Location/Qualifiers
     source          1..4945
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    22..46
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(202..1350)
                     /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(1686..2274)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1766..1785)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2445..3236,3237..3305))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     3068..3087
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3306..3410)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      3497..4128
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4220..4244
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
     protein_bind    4399..4420
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4435..4440,4441..4458,4459..4465)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4473..4489
                     /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     4478..4500
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4497..4513
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4497..4513
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4509..4832
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    4526..4582
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(4583..4600)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4583..4599)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4592..4614)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(4808..4827)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gtggtttaaa ctatcagtgt ttgacaggat atattggcgg gtaaacctaa gagaaaagag
       61 cgtttattag aataatcgga tatttaaaag ggcgtgaaaa ggtttatccg ttcgtccatt
      121 tgtatgtgca tgccaaccac agggttcccc agatcaggcg ctggctgctg aacccccagc
      181 cggaactgac cccacaaggc cctagcgttt gcaatgcacc aggtcatcat tgacccaggc
      241 gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct tcgccgacct gctcgcgcca
      301 cttcttcacg cgggtggaat ccgatccgca catgaggcgg aaggtttcca gcttgagcgg
      361 gtacggctcc cggtgcgagc tgaaatagtc gaacatccgt cgggccgtcg gcgacagctt
      421 gcggtacttc tcccatatga atttcgtgta gtggtcgcca gcaaacagca cgacgatttc
      481 ctcgtcgatc aggacctggc aacgggacgt tttcttgcca cggtccagga cgcggaagcg
      541 gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg gacgtgaagc ccatcgccgt
      601 cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa taccggccat tgatcgacca
      661 gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatc ggctcgccga taggggtgcg
      721 cttcgcgtac tccaacacct gctgccacac cagttcgtca tcgtcggccc gcagctcgac
      781 gccggtgtag gtgatcttca cgtccttgtt gacgtggaaa atgaccttgt tttgcagcgc
      841 ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca gagcgggccg tgtcgtttgg
      901 catcgctcgc atcgtgtccg gccacggcgc aatatcgaac aaggaaagct gcatttcctt
      961 gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttg gcctcgctga cctgttttgc
     1021 caggtcctcg ccggcggttt ttcgcttctt ggtcgtcata gttcctcgcg tgtcgatggt
     1081 catcgacttc gccaaacctg ccgcctcctg ttcaagacga cgcgaacgct ccacggcggc
     1141 cgatggcgcg ggcagggcag ggggagccag ttgcacgctg tcgcgctcga tcttggccgt
     1201 agcttgctgg accatcgagc cgacggactg gaaggtttcg cggggcgcac gcatgacggt
     1261 gcggcttgcg atggtttcgg catcctcggc ggaaaacccc gcgtcgatca gttcttgcct
     1321 gtatgccttc cggtcaaacg tccgattcat tcaccctcct tgcgggattg ccccgactca
     1381 cgccggggca atgtgccctt attcctgatt tgacccgcct ggtgccttgg tgtccagata
     1441 atccacctta tcggcaatga agtcggtccc gtagaccgtc tggccgtcct tctcgtactt
     1501 ggtattccga atcttgccct gcacgaatac cagcgacccc ttgcccaaat acttgccgtg
     1561 ggcctcggcc tgagagccaa aacacttgat gcggaagaag tcggtgcgct cctgcttgtc
     1621 gccggcatcg ttgcgccaca tctaggatct gccaggaacc gtaaaaaggc cgcgttgctg
     1681 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     1741 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1801 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1861 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     1921 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1981 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     2041 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     2101 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     2161 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     2221 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     2281 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     2341 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     2401 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2461 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2521 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2581 ccgcgagaac cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2641 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     2701 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     2761 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2821 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     2881 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     2941 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     3001 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     3061 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     3121 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     3181 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     3241 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     3301 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     3361 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacgaattgg
     3421 ccagcgctgc catttttggg gtgaggccgt tcgcggccga ggggcgcagc ccctgggggg
     3481 atgggaggcc cgcgttagcg ggccgggagg gttcgagaag ggggggcacc ccccttcggc
     3541 gtgcgcggtc acgcgcacag ggcgcagccc tggttaaaaa caaggtttat aaatattggt
     3601 ttaaaagcag gttaaaagac aggttagcgg tggccgaaaa acgggcggaa acccttgcaa
     3661 atgctggatt ttctgcctgt ggacagcccc tcaaatgtca ataggtgcgc ccctcatctg
     3721 tcagcactct gcccctcaag tgtcaaggat cgcgcccctc atctgtcagt agtcgcgccc
     3781 ctcaagtgtc aataccgcag ggcacttatc cccaggcttg tccacatcat ctgtgggaaa
     3841 ctcgcgtaaa atcaggcgtt ttcgccgatt tgcgaggctg gccagctcca cgtcgccggc
     3901 cgaaatcgag cctgcccctc atctgtcaac gccgcgccgg gtgagtcggc ccctcaagtg
     3961 tcaacgtccg cccctcatct gtcagtgagg gccaagtttt ccgcgaggta tccacaacgc
     4021 cggcggccgc ggtgtctcgc acacggcttc gacggcgttt ctggcgcgtt tgcagggcca
     4081 tagacggccg ccagcccagc ggcgagggca accagcccgg tgagcgtcgc aaaggagatc
     4141 ctgatctgac tgatgggctg cctgtatcga gtggtgattt tgtgccgagc tgccggtcgg
     4201 ggagctgttg gctggctggt ggcaggatat attgtggtgt aaacaaattg acgcttagac
     4261 aacttaataa cacattgcgg acgtttttaa tgtactgggg tggatgcagt gggccccact
     4321 ctgtgaagac aattactgag accgcagctg gcacgacagg tttcccgact ggaaagcggg
     4381 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     4441 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     4501 aaacagctat gaccatgatt acgccaagct tgcatgcctg caggtcgact ctagaggatc
     4561 cccgggtacc gagctcgaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     4621 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     4681 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     4741 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atatggtgca
     4801 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     4861 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     4921 cggtctcagg gattgtcttc acgaa
//
