LOCUS       sequence-439147-        6112 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 6112)
  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 6112)
  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: POC1483
FEATURES             Location/Qualifiers
     source          1..6112
                     /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"
     CDS             complement(990..1007)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1017..1034)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1053..1075
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1090..1114
                     /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(1114..1133)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1115..1133)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1217..1236)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1442..1519
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1520..2602
                     /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(1539..1558)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3411..3602
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3596..3618)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3704..3843
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3759..3778
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3858..3875)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4029..4617)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4109..4128)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4728..4747)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4739..5554
                     /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             4739..5554
                     /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(4812..4831)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5646..6101)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5783..5804)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5995..6014
                     /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 cgacggagct cgaattcgga tccttattca cactcgttat agcggaacat ttccagtgat
      241 ccgtgcacat actctgcgtg ggactcacag ccgataaaat tacgctccaa gctttttgcc
      301 actaaagaag tcatcccgga gcccgaaaac aagtccaaga taaggtcatt tttgtgtgat
      361 gatgccttga tcatgcgctc aatcagtgcc ttcggtttga tcgatgggtg tttcggtttc
      421 agaatttttc ctttttcttt ctctacgtgg cgctgcgaag taatttccca cacgtccagg
      481 cacagttttc ccttgggatt ggggaaccaa cgtttattat ttttaaggat acccttggac
      541 tgagcatgtt taatacgttc cgcactctca tacgcaatac gaatttcgtc cgcattaaat
      601 gtgtaatttt tcttgtgcat tgagtaaaac agaatcgatt cttgagcatg gttgtaacgt
      661 ttcttggcat tggcaaaccc gtcttttttc acccatgtaa taaagttcaa gaaatgcact
      721 tttttatgat gtaagtaggc taaaaaaagg gcacagttga aaggggtatt gaaaatatag
      781 aacgatcctg tatctttcag ctttggaagc atcttatcga tccatgcata gctaaaagtt
      841 aagaactctt catcgttttt aaaggagtcc catgatgcaa ttttaagatt atacggtgga
      901 tcgatgatag caagatccac cgacttatcc tcaagcttgt ccaggaaggt gaacacatcc
      961 tcaatgtaga tcttattcag aatcatatgg ctgccgcgcg gcaccaggcc gctgctgtga
     1021 tgatgatgat gatggctgct gcccatggta tatctccttc ttaaagttaa acaaaattat
     1081 ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc
     1141 gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca
     1201 ggtgcggttg ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac
     1261 ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga
     1321 ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc
     1381 aacctactac tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgagatccc
     1441 ggacaccatc gaatggcgca aaacctttcg cggtatggca tgatagcgcc cggaagagag
     1501 tcaattcagg gtggtgaatg tgaaaccagt aacgttatac gatgtcgcag agtatgccgg
     1561 tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc agccacgttt ctgcgaaaac
     1621 gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac attcccaacc gcgtggcaca
     1681 acaactggcg ggcaaacagt cgttgctgat tggcgttgcc acctccagtc tggccctgca
     1741 cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc gatcaactgg gtgccagcgt
     1801 ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt aaagcggcgg tgcacaatct
     1861 tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg ctggatgacc aggatgccat
     1921 tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt cttgatgtct ctgaccagac
     1981 acccatcaac agtattattt tctcccatga agacggtacg cgactgggcg tggagcatct
     2041 ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc ccattaagtt ctgtctcggc
     2101 gcgtctgcgt ctggctggct ggcataaata tctcactcgc aatcaaattc agccgatagc
     2161 ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa caaaccatgc aaatgctgaa
     2221 tgagggcatc gttcccactg cgatgctggt tgccaacgat cagatggcgc tgggcgcaat
     2281 gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat atctcggtag tgggatacga
     2341 cgataccgaa gacagctcat gttatatccc gccgttaacc accatcaaac aggattttcg
     2401 cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc aggcggtgaa
     2461 gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccctgg cgcccaatac
     2521 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc
     2581 ccgactggaa agcgggcagt gagcgcaacg caattaatgt aagttagctc actcattagg
     2641 caccgggatc tcgaccgatg cccttgagag ccttcaaccc agtcagctcc ttccggtggg
     2701 cgcggggcat gactatcgtc gccgcactta tgactgtctt ctttatcatg caactcgtag
     2761 gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga ccgctttcgc tggagcgcga
     2821 cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca cgccctcgct caagccttcg
     2881 tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc cattatcgcc ggcatggcgg
     2941 ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac ccggctaggc tggcggggtt
     3001 gccttactgg ttagcagaat gaatcaccga tacgcgagcg aacgtgaagc gactgctgct
     3061 gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt cggtttccgt gtttcgtaaa
     3121 gtctggaaac gcggaagtca gcgccctgca ccattatgtt ccggatctgc atcgcaggat
     3181 gctgctggct accctgtgga acacctacat ctgtattaac gaagcgctgg cattgaccct
     3241 gagtgatttt tctctggtcc cgccgcatcc ataccgccag ttgtttaccc tcacaacgtt
     3301 ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt gagcatcctc tctcgtttca
     3361 tcggtatcat tacccccatg aacagaaatc ccccttacac ggaggcatca gtgaccaaac
     3421 aggaaaaaac cgcccttaac atggcccgct ttatcagaag ccagacatta acgcttctgg
     3481 agaaactcaa cgagctggac gcggatgaac aggcagacat ctgtgaatcg cttcacgacc
     3541 acgctgatga gctttaccgc agctgcctcg cgcgtttcgg tgatgacggt gaaaacctct
     3601 gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc gggagcagac
     3661 aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc atgacccagt
     3721 cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc agattgtact
     3781 gagagtgcac cattgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
     3841 aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     3901 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     3961 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     4021 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     4081 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     4141 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     4201 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     4261 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     4321 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     4381 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     4441 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     4501 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     4561 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     4621 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     4681 attttggtca tgaacaataa aactgtctgc ttacataaac agtaatacaa ggggtgttat
     4741 gagccatatt caacgggaaa cgtcttgctc taggccgcga ttaaattcca acatggatgc
     4801 tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg cgacaatcta
     4861 tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca aaggtagcgt
     4921 tgccaatgat gttacagatg agatggtcag actaaactgg ctgacggaat ttatgcctct
     4981 tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca ccactgcgat
     5041 ccccgggaaa acagcattcc aggtattaga agaatatcct gattcaggtg aaaatattgt
     5101 tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta attgtccttt
     5161 taacagcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata acggtttggt
     5221 tgatgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag tctggaaaga
     5281 aatgcataaa cttttgccat tctcaccgga ttcagtcgtc actcatggtg atttctcact
     5341 tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg gacgagtcgg
     5401 aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg agttttctcc
     5461 ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata tgaataaatt
     5521 gcagtttcat ttgatgctcg atgagttttt ctaagaatta attcatgagc ggatacatat
     5581 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5641 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     5701 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     5761 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     5821 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     5881 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     5941 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     6001 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     6061 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc ca
//
