LOCUS       sequence-437764-        4949 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Testing the activity of BpiI-associated switch methylases.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4949)
  AUTHORS   Flores-Fernandez CN, Lin D, Robins K, O'Callaghan CA
  TITLE     DNA methylases for site-selective inhibition of type IIS restriction
            enzyme activity.
  JOURNAL   Appl Microbiol Biotechnol. 2024 Jan 25;108(1):174. doi: 
            10.1007/s00253-024-13015-7.
  PUBMED    38270650
REFERENCE   2  (bases 1 to 4949)
  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: "Appl 
            Microbiol Biotechnol."; date: "2024-01-25"; volume: "108(1):174. 
            doi"; pages: " 10.1007/s00253-024-13015-7"
COMMENT     Sequence Label: POC1400
FEATURES             Location/Qualifiers
     source          1..4949
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    84..105
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(120..125,126..143,144..150)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    158..174
                     /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     163..185
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     182..198
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     182..198
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             194..517
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    211..267
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(268..285)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(268..284)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(277..299)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(493..512)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    656..680
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(836..1984)
                     /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(2320..2908)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2400..2419)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3376..3754)
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene; 
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     primer_bind     3701..3720
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3939..4043)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4130..4761
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4853..4877
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 cactctgtga agacgctgcc gcgtcttcgc agctggcacg acaggtttcc cgactggaaa
       61 gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct
      121 ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac
      181 acaggaaaca gctatgacca tgattacgcc aagcttgcat gcctgcaggt cgactctaga
      241 ggatccccgg gtaccgagct cgaattcact ggccgtcgtt ttacaacgtc gtgactggga
      301 aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg
      361 taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga
      421 atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg
      481 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
      541 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
      601 tgtgacttgt cttctgagac ctgaagagca cgaagtggtt taaactatca gtgtttgaca
      661 ggatatattg gcgggtaaac ctaagagaaa agagcgttta ttagaataat cggatattta
      721 aaagggcgtg aaaaggttta tccgttcgtc catttgtatg tgcatgccaa ccacagggtt
      781 ccccagatca ggcgctggct gctgaacccc cagccggaac tgaccccaca aggccctagc
      841 gtttgcaatg caccaggtca tcattgaccc aggcgtgttc caccaggccg ctgcctcgca
      901 actcttcgca ggcttcgccg acctgctcgc gccacttctt cacgcgggtg gaatccgatc
      961 cgcacatgag gcggaaggtt tccagcttga gcgggtacgg ctcccggtgc gagctgaaat
     1021 agtcgaacat ccgtcgggcc gtcggcgaca gcttgcggta cttctcccat atgaatttcg
     1081 tgtagtggtc gccagcaaac agcacgacga tttcctcgtc gatcaggacc tggcaacggg
     1141 acgttttctt gccacggtcc aggacgcgga agcggtgcag cagcgacacc gattccaggt
     1201 gcccaacgcg gtcggacgtg aagcccatcg ccgtcgcctg taggcgcgac aggcattcct
     1261 cggccttcgt gtaataccgg ccattgatcg accagcccag gtcctggcaa agctcgtaga
     1321 acgtgaaggt gatcggctcg ccgatagggg tgcgcttcgc gtactccaac acctgctgcc
     1381 acaccagttc gtcatcgtcg gcccgcagct cgacgccggt gtaggtgatc ttcacgtcct
     1441 tgttgacgtg gaaaatgacc ttgttttgca gcgcctcgcg cgggattttc ttgttgcgcg
     1501 tggtgaacag ggcagagcgg gccgtgtcgt ttggcatcgc tcgcatcgtg tccggccacg
     1561 gcgcaatatc gaacaaggaa agctgcattt ccttgatctg ctgcttcgtg tgtttcagca
     1621 acgcggcctg cttggcctcg ctgacctgtt ttgccaggtc ctcgccggcg gtttttcgct
     1681 tcttggtcgt catagttcct cgcgtgtcga tggtcatcga cttcgccaaa cctgccgcct
     1741 cctgttcaag acgacgcgaa cgctccacgg cggccgatgg cgcgggcagg gcagggggag
     1801 ccagttgcac gctgtcgcgc tcgatcttgg ccgtagcttg ctggaccatc gagccgacgg
     1861 actggaaggt ttcgcggggc gcacgcatga cggtgcggct tgcgatggtt tcggcatcct
     1921 cggcggaaaa ccccgcgtcg atcagttctt gcctgtatgc cttccggtca aacgtccgat
     1981 tcattcaccc tccttgcggg attgccccga ctcacgccgg ggcaatgtgc ccttattcct
     2041 gatttgaccc gcctggtgcc ttggtgtcca gataatccac cttatcggca atgaagtcgg
     2101 tcccgtagac cgtctggccg tccttctcgt acttggtatt ccgaatcttg ccctgcacga
     2161 ataccagcga ccccttgccc aaatacttgc cgtgggcctc ggcctgagag ccaaaacact
     2221 tgatgcggaa gaagtcggtg cgctcctgct tgtcgccggc atcgttgcgc cacatctagg
     2281 atctgccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     2341 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     2401 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     2461 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     2521 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     2581 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     2641 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     2701 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     2761 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     2821 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     2881 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     2941 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     3001 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     3061 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     3121 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     3181 gggcttacca tctggcccca gtgctgcaat gataccgcga gaccacgctc accggctcca
     3241 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3301 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3361 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3421 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3481 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3541 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3601 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     3661 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     3721 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     3781 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     3841 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     3901 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     3961 tgaagcattt atcaaggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4021 aataaacaaa taggggttcc gcgcacgaat tggccagcgc tgccattttt ggggtgaggc
     4081 cgttcgcggc cgaggggcgc agcccctggg gggatgggag gcccgcgtta gcgggccggg
     4141 agggttcgag aagggggggc accccccttc ggcgtgcgcg gtcacgcgca cagggcgcag
     4201 ccctggttaa aaacaaggtt tataaatatt ggtttaaaag caggttaaaa gacaggttag
     4261 cggtggccga aaaacgggcg gaaacccttg caaatgctgg attttctgcc tgtggacagc
     4321 ccctcaaatg tcaataggtg cgcccctcat ctgtcagcac tctgcccctc aagtgtcaag
     4381 gatcgcgccc ctcatctgtc agtagtcgcg cccctcaagt gtcaataccg cagggcactt
     4441 atccccaggc ttgtccacat catctgtggg aaactcgcgt aaaatcaggc gttttcgccg
     4501 atttgcgagg ctggccagct ccacgtcgcc ggccgaaatc gagcctgccc ctcatctgtc
     4561 aacgccgcgc cgggtgagtc ggcccctcaa gtgtcaacgt ccgcccctca tctgtcagtg
     4621 agggccaagt tttccgcgag gtatccacaa cgccggcggc cgcggtgtct cgcacacggc
     4681 ttcgacggcg tttctggcgc gtttgcaggg ccatagacgg ccgccagccc agcggcgagg
     4741 gcaaccagcc cggtgagcgt cgcaaaggag atcctgatct gactgatggg ctgcctgtat
     4801 cgagtggtga ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga
     4861 tatattgtgg tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt
     4921 taatgtactg gggtggatgc agtgggccc
//
