LOCUS       sequence-439178-        6041 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BpiI-associated non-switchable methylase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6041)
  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 6041)
  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: POC1488
FEATURES             Location/Qualifiers
     source          1..6041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             978..1000
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1015..1039
                     /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     complement(1039..1058)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1040..1058)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1146..1165)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1371..1448
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1449..2531
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1468..1487)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3340..3531
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3525..3547)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3633..3772
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3688..3707
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3787..3804)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3958..4546)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4038..4057)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4657..4676)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4668..5483
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4668..5483
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4741..4760)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5575..6030)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5712..5733)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5924..5943
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt
      181 taatctccaa ctggttcagc acaaaatttg cctgatttac atattctgcg ttcatatcgc
      241 atccgatgaa gttacgcccc aacttcttgg ctacaatggc agttgtgcca gaccccatga
      301 aacagtctaa gacaaggtca ttaggattag aactggcgcg aatgatacgt tcaatcaggt
      361 cacggggctt aggggtaatg tgcgtaagtt taacggtttt accattaacc ttctccttgt
      421 gacgctgact actgaaatgc cacacctctc cgcacagacg gccgtttggg ttcgggaacc
      481 aacgttttcc gtttttcagg attcccttct cactggcgtg cttgatacgg tcggttgact
      541 cgtaagggac gcggacctca tcgtagttaa atgtatggtt tttggatttg ctaaagaaca
      601 ggatagtctc ttggcctgtg ctaaaacggc gtttcgctga ccccattccg tcacgtttat
      661 cccacgtaat ccagttttga aagatcattc ccttactaac aaggtactgg caaataaaag
      721 cgcaattaaa cggagtgtta aagatgtaaa gcgacccgtc tttgtccaac ttgtccagaa
      781 ccttatcgat ccagcggtac gtaaatgcca ggaactcgtt atgtgaatca aaagaatccc
      841 aatccgcctt actcaggtta tatggaggat caatgaccgc caactgaaca gatttgttct
      901 caacctggtc caaaaaatcg aagcaattca tttggtggat tttattgatc tctaacatgt
      961 tctctaccat atgtatatct ccttcttaaa gttaaacaaa attatttcta gaggggaatt
     1021 gttatccgct cacaattccc ctatagtgag tcgtattaat ttcgcgggat cgagatcgat
     1081 ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc
     1141 tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat
     1201 gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc
     1261 catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact
     1321 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg
     1381 aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     1441 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
     1501 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
     1561 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
     1621 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     1681 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     1741 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     1801 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     1861 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     1921 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     1981 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     2041 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     2101 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     2161 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     2221 ccgagtccgg gctgcgcgtt ggtgcggaca tctcggtagt gggatacgac gataccgaag
     2281 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
     2341 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     2401 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
     2461 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     2521 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct
     2581 cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg
     2641 actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg
     2701 gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc
     2761 ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc
     2821 gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc cccacgggtg
     2881 cgcatgatcg tgctcctgtc gttgaggacc cggctaggct ggcggggttg ccttactggt
     2941 tagcagaatg aatcaccgat acgcgagcga acgtgaagcg actgctgctg caaaacgtct
     3001 gcgacctgag caacaacatg aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg
     3061 cggaagtcag cgccctgcac cattatgttc cggatctgca tcgcaggatg ctgctggcta
     3121 ccctgtggaa cacctacatc tgtattaacg aagcgctggc attgaccctg agtgattttt
     3181 ctctggtccc gccgcatcca taccgccagt tgtttaccct cacaacgttc cagtaaccgg
     3241 gcatgttcat catcagtaac ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt
     3301 acccccatga acagaaatcc cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc
     3361 gcccttaaca tggcccgctt tatcagaagc cagacattaa cgcttctgga gaaactcaac
     3421 gagctggacg cggatgaaca ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag
     3481 ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag
     3541 ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag
     3601 ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat
     3661 agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc
     3721 attgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgctcttc
     3781 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     3841 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3901 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3961 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4021 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4081 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4141 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4201 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4261 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4321 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4381 ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4441 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4501 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4561 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4621 gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg agccatattc
     4681 aacgggaaac gtcttgctct aggccgcgat taaattccaa catggatgct gatttatatg
     4741 ggtataaatg ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg
     4801 ggaagcccga tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg
     4861 ttacagatga gatggtcaga ctaaactggc tgacggaatt tatgcctctt ccgaccatca
     4921 agcattttat ccgtactcct gatgatgcat ggttactcac cactgcgatc cccgggaaaa
     4981 cagcattcca ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg
     5041 cagtgttcct gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc
     5101 gcgtatttcg tctcgctcag gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg
     5161 attttgatga cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataaac
     5221 ttttgccatt ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta
     5281 tttttgacga ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc
     5341 gataccagga tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga
     5401 aacggctttt tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcatt
     5461 tgatgctcga tgagtttttc taagaattaa ttcatgagcg gatacatatt tgaatgtatt
     5521 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctaaattg
     5581 taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta
     5641 accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt
     5701 tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca
     5761 aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa
     5821 gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat
     5881 ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag
     5941 gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg
     6001 ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc a
//
