LOCUS       sequence-439151-        6023 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 6023)
  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 6023)
  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: POC1484
FEATURES             Location/Qualifiers
     source          1..6023
                     /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             960..982
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    997..1021
                     /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(1021..1040)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1022..1040)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1128..1147)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1353..1430
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1431..2513
                     /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(1450..1469)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3322..3513
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3507..3529)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3615..3754
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3670..3689
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3769..3786)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3940..4528)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4020..4039)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4639..4658)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4650..5465
                     /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             4650..5465
                     /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(4723..4742)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5557..6012)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5694..5715)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5906..5925
                     /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 gcttcacact
      181 cgttatagcg gaacatttcc agtgatccgt gcacatactc tgcgtgggac tcacagccga
      241 taaaattacg ctccaagctt tttgccacta aagaagtcat cccggagccc gaaaacaagt
      301 ccaagataag gtcatttttg tgtgatgatg ccttgatcat gcgctcaatc agtgccttcg
      361 gtttgatcga tgggtgtttc ggtttcagaa tttttccttt ttctttctct acgtggcgct
      421 gcgaagtaat ttcccacacg tccaggcaca gttttccctt gggattgggg aaccaacgtt
      481 tattattttt aaggataccc ttggactgag catgtttaat acgttccgca ctctcatacg
      541 caatacgaat ttcgtccgca ttaaatgtgt aatttttctt gtgcattgag taaaacagaa
      601 tcgattcttg agcatggttg taacgtttct tggcattggc aaacccgtct tttttcaccc
      661 atgtaataaa gttcaagaaa tgcacttttt tatgatgtaa gtaggctaaa aaaagggcac
      721 agttgaaagg ggtattgaaa atatagaacg atcctgtatc tttcagcttt ggaagcatct
      781 tatcgatcca tgcatagcta aaagttaaga actcttcatc gtttttaaag gagtcccatg
      841 atgcaatttt aagattatac ggtggatcga tgatagcaag atccaccgac ttatcctcaa
      901 gcttgtccag gaaggtgaac acatcctcaa tgtagatctt attcagaatc atatgtatat
      961 ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc
     1021 ccctatagtg agtcgtatta atttcgcggg atcgagatcg atctcgatcc tctacgccgg
     1081 acgcatcgtg gccggcatca ccggcgccac aggtgcggtt gctggcgcct atatcgccga
     1141 catcaccgat ggggaagatc gggctcgcca cttcgggctc atgagcgctt gtttcggcgt
     1201 gggtatggtg gcaggccccg tggccggggg actgttgggc gccatctcct tgcatgcacc
     1261 attccttgcg gcggcggtgc tcaacggcct caacctacta ctgggctgct tcctaatgca
     1321 ggagtcgcat aagggagagc gtcgagatcc cggacaccat cgaatggcgc aaaacctttc
     1381 gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat gtgaaaccag
     1441 taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt tcccgcgtgg
     1501 tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg gcgatggcgg
     1561 agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag tcgttgctga
     1621 ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc gcggcgatta
     1681 aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa cgaagcggcg
     1741 tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt gggctgatca
     1801 ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc actaatgttc
     1861 cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt ttctcccatg
     1921 aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag caaatcgcgc
     1981 tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc tggcataaat
     2041 atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg agtgccatgt
     2101 ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact gcgatgctgg
     2161 ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc gggctgcgcg
     2221 ttggtgcgga catctcggta gtgggatacg acgataccga agacagctca tgttatatcc
     2281 cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc gtggaccgct
     2341 tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc gtctcactgg
     2401 tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     2461 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     2521 gcaattaatg taagttagct cactcattag gcaccgggat ctcgaccgat gcccttgaga
     2581 gccttcaacc cagtcagctc cttccggtgg gcgcggggca tgactatcgt cgccgcactt
     2641 atgactgtct tctttatcat gcaactcgta ggacaggtgc cggcagcgct ctgggtcatt
     2701 ttcggcgagg accgctttcg ctggagcgcg acgatgatcg gcctgtcgct tgcggtattc
     2761 ggaatcttgc acgccctcgc tcaagccttc gtcactggtc ccgccaccaa acgtttcggc
     2821 gagaagcagg ccattatcgc cggcatggcg gccccacggg tgcgcatgat cgtgctcctg
     2881 tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa tgaatcaccg
     2941 atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg agcaacaaca
     3001 tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc agcgccctgc
     3061 accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg aacacctaca
     3121 tctgtattaa cgaagcgctg gcattgaccc tgagtgattt ttctctggtc ccgccgcatc
     3181 cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc atcatcagta
     3241 acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat gaacagaaat
     3301 cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa catggcccgc
     3361 tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga cgcggatgaa
     3421 caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg cagctgcctc
     3481 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     3541 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     3601 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc
     3661 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccattgcggt gtgaaatacc
     3721 gcacagatgc gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga
     3781 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     3841 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     3901 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     3961 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     4021 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     4081 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     4141 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     4201 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     4261 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     4321 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     4381 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     4441 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     4501 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     4561 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgaacaata aaactgtctg
     4621 cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa acgtcttgct
     4681 ctaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg
     4741 ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag
     4801 agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca
     4861 gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc
     4921 ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc caggtattag
     4981 aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt
     5041 tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt cgtctcgctc
     5101 aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat gacgagcgta
     5161 atggctggcc tgttgaacaa gtctggaaag aaatgcataa acttttgcca ttctcaccgg
     5221 attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat
     5281 taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca
     5341 tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat
     5401 atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc gatgagtttt
     5461 tctaagaatt aattcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5521 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     5581 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     5641 gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt
     5701 ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct
     5761 atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt
     5821 gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa
     5881 agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc
     5941 tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc
     6001 tacagggcgc gtcccattcg cca
//
