LOCUS       sequence-437748-        4958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Testing the activity of BsaI-associated switch methylases.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4958)
  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 4958)
  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: POC1399
FEATURES             Location/Qualifiers
     source          1..4958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    92..113
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(128..133,134..151,152..158)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    166..182
                     /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     171..193
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     190..206
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     190..206
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             202..525
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    219..275
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(276..293)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(276..292)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(285..307)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(501..520)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_feature    664..688
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     CDS             complement(844..1992)
                     /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(2328..2916)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2408..2427)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3087..3878,3879..3947))
                     /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     3710..3729
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3948..4052)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4139..4770
                     /label=oriV
                     /note="incP origin of replication"
     misc_feature    4862..4886
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA"
ORIGIN
        1 cactctgtga cttggtctca tgcctgagac caagtcgcag ctggcacgac aggtttcccg
       61 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
      121 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
      181 aatttcacac aggaaacagc tatgaccatg attacgccaa gcttgcatgc ctgcaggtcg
      241 actctagagg atccccgggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt
      301 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
      361 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
      421 aatggcgaat ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
      481 cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga
      541 cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac
      601 agacaagctg tgacttgtct tctgagacct gaagagcacg aagtggttta aactatcagt
      661 gtttgacagg atatattggc gggtaaacct aagagaaaag agcgtttatt agaataatcg
      721 gatatttaaa agggcgtgaa aaggtttatc cgttcgtcca tttgtatgtg catgccaacc
      781 acagggttcc ccagatcagg cgctggctgc tgaaccccca gccggaactg accccacaag
      841 gccctagcgt ttgcaatgca ccaggtcatc attgacccag gcgtgttcca ccaggccgct
      901 gcctcgcaac tcttcgcagg cttcgccgac ctgctcgcgc cacttcttca cgcgggtgga
      961 atccgatccg cacatgaggc ggaaggtttc cagcttgagc gggtacggct cccggtgcga
     1021 gctgaaatag tcgaacatcc gtcgggccgt cggcgacagc ttgcggtact tctcccatat
     1081 gaatttcgtg tagtggtcgc cagcaaacag cacgacgatt tcctcgtcga tcaggacctg
     1141 gcaacgggac gttttcttgc cacggtccag gacgcggaag cggtgcagca gcgacaccga
     1201 ttccaggtgc ccaacgcggt cggacgtgaa gcccatcgcc gtcgcctgta ggcgcgacag
     1261 gcattcctcg gccttcgtgt aataccggcc attgatcgac cagcccaggt cctggcaaag
     1321 ctcgtagaac gtgaaggtga tcggctcgcc gataggggtg cgcttcgcgt actccaacac
     1381 ctgctgccac accagttcgt catcgtcggc ccgcagctcg acgccggtgt aggtgatctt
     1441 cacgtccttg ttgacgtgga aaatgacctt gttttgcagc gcctcgcgcg ggattttctt
     1501 gttgcgcgtg gtgaacaggg cagagcgggc cgtgtcgttt ggcatcgctc gcatcgtgtc
     1561 cggccacggc gcaatatcga acaaggaaag ctgcatttcc ttgatctgct gcttcgtgtg
     1621 tttcagcaac gcggcctgct tggcctcgct gacctgtttt gccaggtcct cgccggcggt
     1681 ttttcgcttc ttggtcgtca tagttcctcg cgtgtcgatg gtcatcgact tcgccaaacc
     1741 tgccgcctcc tgttcaagac gacgcgaacg ctccacggcg gccgatggcg cgggcagggc
     1801 agggggagcc agttgcacgc tgtcgcgctc gatcttggcc gtagcttgct ggaccatcga
     1861 gccgacggac tggaaggttt cgcggggcgc acgcatgacg gtgcggcttg cgatggtttc
     1921 ggcatcctcg gcggaaaacc ccgcgtcgat cagttcttgc ctgtatgcct tccggtcaaa
     1981 cgtccgattc attcaccctc cttgcgggat tgccccgact cacgccgggg caatgtgccc
     2041 ttattcctga tttgacccgc ctggtgcctt ggtgtccaga taatccacct tatcggcaat
     2101 gaagtcggtc ccgtagaccg tctggccgtc cttctcgtac ttggtattcc gaatcttgcc
     2161 ctgcacgaat accagcgacc ccttgcccaa atacttgccg tgggcctcgg cctgagagcc
     2221 aaaacacttg atgcggaaga agtcggtgcg ctcctgcttg tcgccggcat cgttgcgcca
     2281 catctaggat ctgccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2341 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2401 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2461 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2521 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2581 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2641 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2701 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     2761 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     2821 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     2881 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     2941 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3001 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3061 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3121 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3181 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga accacgctca
     3241 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3301 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3361 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3421 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3481 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3541 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3601 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3661 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3721 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     3781 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     3841 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     3901 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     3961 caatattatt gaagcattta tcaaggttat tgtctcatga gcggatacat atttgaatgt
     4021 atttagaaaa ataaacaaat aggggttccg cgcacgaatt ggccagcgct gccatttttg
     4081 gggtgaggcc gttcgcggcc gaggggcgca gcccctgggg ggatgggagg cccgcgttag
     4141 cgggccggga gggttcgaga agggggggca ccccccttcg gcgtgcgcgg tcacgcgcac
     4201 agggcgcagc cctggttaaa aacaaggttt ataaatattg gtttaaaagc aggttaaaag
     4261 acaggttagc ggtggccgaa aaacgggcgg aaacccttgc aaatgctgga ttttctgcct
     4321 gtggacagcc cctcaaatgt caataggtgc gcccctcatc tgtcagcact ctgcccctca
     4381 agtgtcaagg atcgcgcccc tcatctgtca gtagtcgcgc ccctcaagtg tcaataccgc
     4441 agggcactta tccccaggct tgtccacatc atctgtggga aactcgcgta aaatcaggcg
     4501 ttttcgccga tttgcgaggc tggccagctc cacgtcgccg gccgaaatcg agcctgcccc
     4561 tcatctgtca acgccgcgcc gggtgagtcg gcccctcaag tgtcaacgtc cgcccctcat
     4621 ctgtcagtga gggccaagtt ttccgcgagg tatccacaac gccggcggcc gcggtgtctc
     4681 gcacacggct tcgacggcgt ttctggcgcg tttgcagggc catagacggc cgccagccca
     4741 gcggcgaggg caaccagccc ggtgagcgtc gcaaaggaga tcctgatctg actgatgggc
     4801 tgcctgtatc gagtggtgat tttgtgccga gctgccggtc ggggagctgt tggctggctg
     4861 gtggcaggat atattgtggt gtaaacaaat tgacgcttag acaacttaat aacacattgc
     4921 ggacgttttt aatgtactgg ggtggatgca gtgggccc
//
