LOCUS       sequence-439177-        6979 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  BsaI-associated non-switchable methylase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6979)
  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 6979)
  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: POC1479
FEATURES             Location/Qualifiers
     source          1..6979
                     /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(1857..1874)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1884..1901)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1920..1942
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1957..1981
                     /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(1981..2000)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1982..2000)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2084..2103)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2309..2386
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2387..3469
                     /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(2406..2425)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4278..4469
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4463..4485)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4571..4710
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4626..4645
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4725..4742)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4896..5484)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4976..4995)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5595..5614)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5606..6421
                     /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             5606..6421
                     /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(5679..5698)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6513..6968)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6650..6671)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6862..6881
                     /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 tccttaatcg tagtgctgga agatgcgtaa caaagactct
      241 ttttctgcca actcgatttt gtcgaagcta ctaagcaatt tgttgaactc attcaggtca
      301 agtttgtaaa tgttcttcgc cacatatgct tcaatcttta attccgcatc gcagcacccc
      361 tcaatgcagc taattaccag ctgtttcaac tcactgtgtt gcagtaagat cttttcgcta
      421 gggatcgcgg ttttacgaag agcagacaga ctaacatgcc cagtagccaa aaaggagcgc
      481 aattgaaatt cgaagcacag agagttaata attccaagca aggaaaacag ggaatcctgg
      541 gacccggatt tgtagtatac tacacccaat gagttaccag ccaacgagtt aggcggaatg
      601 atagttgcga tcatgcggcg tttttggctt gggcgcgaaa tatcgcgcca ggcgatgcgg
      661 acgaagttgc tattggaaag caccttgtca atctttttcg taatgcgcag agacttttca
      721 ttgtggaaat tgtagcggcc aatcattttg cccttaataa acaacagccc cccacccgac
      781 ttctgagtcc aatccgacga gccagtttcg tcgatttcac ggcccgccca aatctcgccg
      841 ttcttttcta aactacccca cgttgggtaa ccctcttgaa gtttgcgcaa aagatggacc
      901 gcatgcacgc cttgagacac ggggatcgac agatattggg aatcgatgct tgacaaatcg
      961 aaaaaaccca atgacttgat gttcaaattt ttatcgtaga cgcttaattg aatattatca
     1021 ttcaaggatt ctcccttgtt aaaaatgaag gagcaggagc tcacatctgc tccaccgaaa
     1081 agttttgctt cggctggata atagttaata cagtcgatac gtccggaatt gaaaaacttc
     1141 tcacgtaaaa tataggattg ctcgtccgca aaaaagctgg cgggcatgac gatcgctact
     1201 aacccatttt tgttgcaaat ctcaagagat aactctgcac caacacgact taagttcgta
     1261 ccccagccag cgaacttgcg cttcggctga ctatagggaa gcacacgtga aaggtaatca
     1321 tcaaattcac gaaggctgtc aatatacatg gatttcatgc ttggttcgaa aaaatcaagc
     1381 tcacgagaat caggtttaat gttctcccag ggaggatttg tgatcacaca gtcgaagtag
     1441 tctacatact tcttaaattc gtaaaataca tccgagttct taattgtaat attgatatta
     1501 gcgccggcta acttcaattc cgacagagcg tttttggcgt aagtcagacc ggtgttctca
     1561 atgtcaaaca ggtagacgtt ccattcgaca tcggggaagc cgttaatcat ccactgcttg
     1621 atcaaaagag tgatcagacg accgtcacct gaaaagggat cagcaacgtt atagctcttt
     1681 ttatcacagg agttaatcag cggaagtaac ttctcgatca ttaacagcgc gattttgggg
     1741 tcggtgtagt atttaccggt agcttttttc gtgaacttag tagtttcgtc cagcgacagg
     1801 ctgttgatta aatggatgtg tttcatatag aagatctcct ccatgctagc catatggctg
     1861 ccgcgcggca ccaggccgct gctgtgatga tgatgatgat ggctgctgcc catggtatat
     1921 ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg ctcacaattc
     1981 ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta cgccggacgc
     2041 atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc
     2101 accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt
     2161 atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca tgcaccattc
     2221 cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct aatgcaggag
     2281 tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa cctttcgcgg
     2341 tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga aaccagtaac
     2401 gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc gcgtggtgaa
     2461 ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga tggcggagct
     2521 gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt tgctgattgg
     2581 cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg cgattaaatc
     2641 tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa gcggcgtcga
     2701 agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc tgatcattaa
     2761 ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta atgttccggc
     2821 gttatttctt gatgtctctg accagacacc catcaacagt attattttct cccatgaaga
     2881 cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa tcgcgctgtt
     2941 agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc ataaatatct
     3001 cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg ccatgtccgg
     3061 ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga tgctggttgc
     3121 caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc tgcgcgttgg
     3181 tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt atatcccgcc
     3241 gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg accgcttgct
     3301 gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct cactggtgaa
     3361 aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc
     3421 attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa
     3481 ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc ttgagagcct
     3541 tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga
     3601 ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg gtcattttcg
     3661 gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg gtattcggaa
     3721 tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt ttcggcgaga
     3781 agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg ctcctgtcgt
     3841 tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac
     3901 gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa
     3961 tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca
     4021 ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg
     4081 tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata
     4141 ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc
     4201 gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc
     4261 cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta
     4321 tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg
     4381 cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc
     4441 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     4501 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     4561 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     4621 ctatgcggca tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac
     4681 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     4741 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4801 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4861 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4921 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4981 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5041 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5101 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5161 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5221 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5281 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     5341 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5401 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5461 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5521 cagtggaacg aaaactcacg ttaagggatt ttggtcatga acaataaaac tgtctgctta
     5581 cataaacagt aatacaaggg gtgttatgag ccatattcaa cgggaaacgt cttgctctag
     5641 gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg ctcgcgataa
     5701 tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg cgccagagtt
     5761 gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga tggtcagact
     5821 aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc gtactcctga
     5881 tgatgcatgg ttactcacca ctgcgatccc cgggaaaaca gcattccagg tattagaaga
     5941 atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc gccggttgca
     6001 ttcgattcct gtttgtaatt gtccttttaa cagcgatcgc gtatttcgtc tcgctcaggc
     6061 gcaatcacga atgaataacg gtttggttga tgcgagtgat tttgatgacg agcgtaatgg
     6121 ctggcctgtt gaacaagtct ggaaagaaat gcataaactt ttgccattct caccggattc
     6181 agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg ggaaattaat
     6241 aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc ttgccatcct
     6301 atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc aaaaatatgg
     6361 tattgataat cctgatatga ataaattgca gtttcatttg atgctcgatg agtttttcta
     6421 agaattaatt catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6481 ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa
     6541 attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa
     6601 aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa
     6661 caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca
     6721 gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg
     6781 taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc
     6841 ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc
     6901 aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca
     6961 gggcgcgtcc cattcgcca
//
