LOCUS       sequence-442885-        6104 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 6104)
  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 6104)
  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: POC1486
FEATURES             Location/Qualifiers
     source          1..6104
                     /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             1041..1063
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1078..1102
                     /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(1102..1121)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1103..1121)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1209..1228)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1434..1511
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1512..2594
                     /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(1531..1550)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3403..3594
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3588..3610)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3696..3835
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3751..3770
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3850..3867)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4021..4609)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4101..4120)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4720..4739)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4731..5546
                     /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             4731..5546
                     /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(4804..4823)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5638..6093)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5775..5796)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5987..6006
                     /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 gcttcaaagt
      181 caaataaatt gcccaatttc tgctcgatca gcggtttata gttttcatac agctcgtacc
      241 caatgaagtt gcgtttaaga agcttggctt cgcgaagcgt cgtgccggag cccgaaaatg
      301 ggtctaagac gacgtcgccg acacaagaat aaagacgaat caagcggcgg gccagttctg
      361 cgggcatcag ggctgcgtgt tttccaaaag cgatgtcgtt cttattagga ataggaatct
      421 cccagatttg cttagtgaac tctacccatt cctcttgggt taattgactt tgctcttttt
      481 gctcttcagt aggttgtttt ggtttaccat ccttcacgaa aacgccgatg aactcaatag
      541 tattctgtgc gtaaaaattg cgggggtacg ggtaggatcc aaacatcaag cgtttcgttg
      601 gattggcacg cttccaaata tacacatcca acaaaaacat cttcggctta tttttcaaca
      661 tgttgtttaa atcatgaaga attgagtgtt gaatatccgc gtgtaagtcg aaaatgtgac
      721 gattataatg cgtattcaat accttcttca acataggcat caatggtacg ttgatgcaca
      781 gtttaccgtt tggcttcaga gcacgataac attccaacca aaccttcaac agacccagca
      841 gataatcctc gtatttctct aaagcaccca aatcttccac atgctgagca gaatgttgca
      901 agtcttgtgt gccatttttg gcatagtctt tgatattaaa ataaggaggc gaggtgatga
      961 taagatcaac cgaattatcg ggtacttcgt tcatatttgt cgatgaatgg tagaacacct
     1021 tattaatgtt catatgtata tctccttctt aaagttaaac aaaattattt ctagagggga
     1081 attgttatcc gctcacaatt cccctatagt gagtcgtatt aatttcgcgg gatcgagatc
     1141 gatctcgatc ctctacgccg gacgcatcgt ggccggcatc accggcgcca caggtgcggt
     1201 tgctggcgcc tatatcgccg acatcaccga tggggaagat cgggctcgcc acttcgggct
     1261 catgagcgct tgtttcggcg tgggtatggt ggcaggcccc gtggccgggg gactgttggg
     1321 cgccatctcc ttgcatgcac cattccttgc ggcggcggtg ctcaacggcc tcaacctact
     1381 actgggctgc ttcctaatgc aggagtcgca taagggagag cgtcgagatc ccggacacca
     1441 tcgaatggcg caaaaccttt cgcggtatgg catgatagcg cccggaagag agtcaattca
     1501 gggtggtgaa tgtgaaacca gtaacgttat acgatgtcgc agagtatgcc ggtgtctctt
     1561 atcagaccgt ttcccgcgtg gtgaaccagg ccagccacgt ttctgcgaaa acgcgggaaa
     1621 aagtggaagc ggcgatggcg gagctgaatt acattcccaa ccgcgtggca caacaactgg
     1681 cgggcaaaca gtcgttgctg attggcgttg ccacctccag tctggccctg cacgcgccgt
     1741 cgcaaattgt cgcggcgatt aaatctcgcg ccgatcaact gggtgccagc gtggtggtgt
     1801 cgatggtaga acgaagcggc gtcgaagcct gtaaagcggc ggtgcacaat cttctcgcgc
     1861 aacgcgtcag tgggctgatc attaactatc cgctggatga ccaggatgcc attgctgtgg
     1921 aagctgcctg cactaatgtt ccggcgttat ttcttgatgt ctctgaccag acacccatca
     1981 acagtattat tttctcccat gaagacggta cgcgactggg cgtggagcat ctggtcgcat
     2041 tgggtcacca gcaaatcgcg ctgttagcgg gcccattaag ttctgtctcg gcgcgtctgc
     2101 gtctggctgg ctggcataaa tatctcactc gcaatcaaat tcagccgata gcggaacggg
     2161 aaggcgactg gagtgccatg tccggttttc aacaaaccat gcaaatgctg aatgagggca
     2221 tcgttcccac tgcgatgctg gttgccaacg atcagatggc gctgggcgca atgcgcgcca
     2281 ttaccgagtc cgggctgcgc gttggtgcgg acatctcggt agtgggatac gacgataccg
     2341 aagacagctc atgttatatc ccgccgttaa ccaccatcaa acaggatttt cgcctgctgg
     2401 ggcaaaccag cgtggaccgc ttgctgcaac tctctcaggg ccaggcggtg aagggcaatc
     2461 agctgttgcc cgtctcactg gtgaaaagaa aaaccaccct ggcgcccaat acgcaaaccg
     2521 cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg
     2581 aaagcgggca gtgagcgcaa cgcaattaat gtaagttagc tcactcatta ggcaccggga
     2641 tctcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg ggcgcggggc
     2701 atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt aggacaggtg
     2761 ccggcagcgc tctgggtcat tttcggcgag gaccgctttc gctggagcgc gacgatgatc
     2821 ggcctgtcgc ttgcggtatt cggaatcttg cacgccctcg ctcaagcctt cgtcactggt
     2881 cccgccacca aacgtttcgg cgagaagcag gccattatcg ccggcatggc ggccccacgg
     2941 gtgcgcatga tcgtgctcct gtcgttgagg acccggctag gctggcgggg ttgccttact
     3001 ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa gcgactgctg ctgcaaaacg
     3061 tctgcgacct gagcaacaac atgaatggtc ttcggtttcc gtgtttcgta aagtctggaa
     3121 acgcggaagt cagcgccctg caccattatg ttccggatct gcatcgcagg atgctgctgg
     3181 ctaccctgtg gaacacctac atctgtatta acgaagcgct ggcattgacc ctgagtgatt
     3241 tttctctggt cccgccgcat ccataccgcc agttgtttac cctcacaacg ttccagtaac
     3301 cgggcatgtt catcatcagt aacccgtatc gtgagcatcc tctctcgttt catcggtatc
     3361 attaccccca tgaacagaaa tcccccttac acggaggcat cagtgaccaa acaggaaaaa
     3421 accgccctta acatggcccg ctttatcaga agccagacat taacgcttct ggagaaactc
     3481 aacgagctgg acgcggatga acaggcagac atctgtgaat cgcttcacga ccacgctgat
     3541 gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg
     3601 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt
     3661 cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc
     3721 gatagcggag tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
     3781 accattgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc
     3841 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3901 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3961 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     4021 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     4081 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4141 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4201 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4261 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4321 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4381 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4441 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     4501 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4561 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4621 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4681 catgaacaat aaaactgtct gcttacataa acagtaatac aaggggtgtt atgagccata
     4741 ttcaacggga aacgtcttgc tctaggccgc gattaaattc caacatggat gctgatttat
     4801 atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt
     4861 atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg
     4921 atgttacaga tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca
     4981 tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atccccggga
     5041 aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc
     5101 tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg
     5161 atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga
     5221 gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata
     5281 aacttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc
     5341 ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag
     5401 accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac
     5461 agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc
     5521 atttgatgct cgatgagttt ttctaagaat taattcatga gcggatacat atttgaatgt
     5581 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctaaa
     5641 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     5701 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     5761 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     5821 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     5881 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     5941 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     6001 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     6061 ccgccgcgct taatgcgccg ctacagggcg cgtcccattc gcca
//
