LOCUS       sequence-439150-        6416 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 6416)
  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 6416)
  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: POC1466
FEATURES             Location/Qualifiers
     source          1..6416
                     /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             1353..1375
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1390..1414
                     /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(1414..1433)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1415..1433)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1521..1540)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1746..1823
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1824..2906
                     /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(1843..1862)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3715..3906
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3900..3922)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4008..4147
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4063..4082
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4162..4179)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4333..4921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4413..4432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5032..5051)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5043..5858
                     /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             5043..5858
                     /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(5116..5135)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5950..6405)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6087..6108)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6299..6318
                     /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 gcaagcttaa
      181 gcgacggctt aatgttgcgt tcataatact ctgagggtgt aatgtttcca cgcataaaca
      241 actggtaatc attttccggg tggtggatat agtatttacc agatccatcc tcgccgtgtt
      301 gtaatggggg aaggtcagat aacgcatctt ttactgacac ccaagactct ttatcttccg
      361 tcagcttctg aggtggttcg atcgagaaca gcggatcatt gctgcccaca agaattacac
      421 gcttacggcg ttgtggaatt gcgtaatgtt ccgaatttaa aatccaacca ttcatggttt
      481 tcattgtgga cgagaattcc tccttaacac gttcaaagac ctttcctttt tccatgttca
      541 acaggccggc gacgttctcg aaaatgaatc cattcggctt aatctcgtta agcagcgact
      601 tgtagtgcat aaacagactg ttgcgtgggt cgtccattgt acgcgcgttg ccagccgtgc
      661 taaatccctg gcacggaggc ccgcccagaa tccacagctg tttatcaaaa tataatttct
      721 taaagccaga aatcttgtct gcgatcttag aaaaagtttc gttgtccgaa atgcttccgc
      781 acaaaacttc tttgtgtacg ttgttcgagt atgtctgtaa gtacttttcc tcaatgtcat
      841 tagctaacaa tggagtccac ccggcccact taaagcccaa ccccaatccg cctgcaccac
      901 tgaataagtc aatgtagtag ccggtattac cgatcgcttt ttcaatttct tttgcaataa
      961 gaaacgcaag aaagggaggc acggcattac cgatttgcgt attaatcgcc gtttgacctc
     1021 caaaaaagat aaagtcatca gggaacgact gcagacgtgc agcttcgcgt tgtgaaagta
     1081 cgcgatcttg ctcgtagtga atgtggcatc cgttgccagg acgattgaaa taagtattga
     1141 tcgtataagc tggcatatcg ggcaataaac gcccgtagta tgtgctgcgc gacccttttc
     1201 cttgagcata gctgtcacga atctgttcga tacgtttcga tggtacatcc aagggaatat
     1261 tcttccagtt tcccccaggc gggacagagt taacgatcat catatccagc tcacttaatt
     1321 tggcggcctt gtggtttgga atcatatgta tatctccttc ttaaagttaa acaaaattat
     1381 ttctagaggg gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc
     1441 gggatcgaga tcgatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc
     1501 cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg
     1561 ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg
     1621 gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg
     1681 cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga
     1741 tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     1801 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     1861 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     1921 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     1981 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     2041 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     2101 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     2161 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     2221 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     2281 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     2341 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     2401 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     2461 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     2521 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     2581 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggacatctcg gtagtgggat
     2641 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     2701 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     2761 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     2821 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     2881 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     2941 taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg
     3001 tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc
     3061 gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc
     3121 gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc
     3181 ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg
     3241 gcggccccac gggtgcgcat gatcgtgctc ctgtcgttga ggacccggct aggctggcgg
     3301 ggttgcctta ctggttagca gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc
     3361 tgctgcaaaa cgtctgcgac ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg
     3421 taaagtctgg aaacgcggaa gtcagcgccc tgcaccatta tgttccggat ctgcatcgca
     3481 ggatgctgct ggctaccctg tggaacacct acatctgtat taacgaagcg ctggcattga
     3541 ccctgagtga tttttctctg gtcccgccgc atccataccg ccagttgttt accctcacaa
     3601 cgttccagta accgggcatg ttcatcatca gtaacccgta tcgtgagcat cctctctcgt
     3661 ttcatcggta tcattacccc catgaacaga aatccccctt acacggaggc atcagtgacc
     3721 aaacaggaaa aaaccgccct taacatggcc cgctttatca gaagccagac attaacgctt
     3781 ctggagaaac tcaacgagct ggacgcggat gaacaggcag acatctgtga atcgcttcac
     3841 gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac
     3901 ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc
     3961 agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc
     4021 cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca gagcagattg
     4081 tactgagagt gcaccattgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     4141 catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4201 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4261 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4321 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4381 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4441 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4501 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4561 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4621 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4681 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4741 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     4801 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4861 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4921 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4981 agggattttg gtcatgaaca ataaaactgt ctgcttacat aaacagtaat acaaggggtg
     5041 ttatgagcca tattcaacgg gaaacgtctt gctctaggcc gcgattaaat tccaacatgg
     5101 atgctgattt atatgggtat aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa
     5161 tctatcgatt gtatgggaag cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta
     5221 gcgttgccaa tgatgttaca gatgagatgg tcagactaaa ctggctgacg gaatttatgc
     5281 ctcttccgac catcaagcat tttatccgta ctcctgatga tgcatggtta ctcaccactg
     5341 cgatccccgg gaaaacagca ttccaggtat tagaagaata tcctgattca ggtgaaaata
     5401 ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc
     5461 cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca atcacgaatg aataacggtt
     5521 tggttgatgc gagtgatttt gatgacgagc gtaatggctg gcctgttgaa caagtctgga
     5581 aagaaatgca taaacttttg ccattctcac cggattcagt cgtcactcat ggtgatttct
     5641 cacttgataa ccttattttt gacgagggga aattaatagg ttgtattgat gttggacgag
     5701 tcggaatcgc agaccgatac caggatcttg ccatcctatg gaactgcctc ggtgagtttt
     5761 ctccttcatt acagaaacgg ctttttcaaa aatatggtat tgataatcct gatatgaata
     5821 aattgcagtt tcatttgatg ctcgatgagt ttttctaaga attaattcat gagcggatac
     5881 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     5941 gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     6001 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     6061 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     6121 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     6181 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     6241 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     6301 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     6361 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgcca
//
