LOCUS       sequence-437780-        6843 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 6843)
  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 6843)
  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: POC1469
FEATURES             Location/Qualifiers
     source          1..6843
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..15,16..32,33..38)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(213..260)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     256..274
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(1474..1491)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1501..1518)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1537..1559
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1574..1598
                     /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(1598..1617)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1599..1617)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1701..1720)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1926..2003
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2004..3086
                     /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(2023..2042)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4398..4589
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4583..4605)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4691..4830
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4746..4765
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4845..4862)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5016..5604)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5096..5115)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5775..6566,6567..6635))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6398..6417
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6636..6740)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6808..6826
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa
       61 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg
      121 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt
      181 gcgggatatc cggatatagt tcctcctttc agcaaaaaac ccctcaagac ccgtttagag
      241 gccccaaggg gttatgctag ttattgctca gcggtggcag cagccaactc agcttccttt
      301 cgggctttgt tagcagccgg atccttaaat gatagctaag gaggacagca tctcgatata
      361 ctgctgggct gtgatcttct tgcgcatgaa tgactgataa atattttgtg gctcacaaac
      421 gtagttcttc caagagccgt cctcattagg cgacaaaagg ggtaagtctg aaagggcctc
      481 ctttacggaa atggcactgc ttaaggcgtc aaacagggtc ttttctcctt taagagacgt
      541 aattgggata gggggctcgc tgttcttcgt tccggtggag tccccgatga tgactacgcg
      601 gttgcggcgt tggggcacac cgtaatccac cgaattaagt ttatatacga aaagattcga
      661 caccgtctta gacaattccg atttaaccat ctcaaagaat gctcccttct ccatcgacaa
      721 caggccagtc acgttttcaa aaataaaacc gtctggcttg acttctttca ggatactcac
      781 gtaactttcg aacagccagt tgcgctcgtc cacgattgaa cgtttttttc ctgcagtcga
      841 aaacccttgg cagggtggtc ccccaagaac aaacaatgga cggtccgggt tcgacttctt
      901 tgcgatcagg cacttttgaa tgatagtctc actgaccttt tcgtcgcgga tatccccaag
      961 cactacttca ttgtgaatat tattcatgta ggtggtcagc gcccatttat cgatgtcatt
     1021 tgcgatgatc ggtttccacc cggcccaaag gaaacctaag gaaaggcccc ccgccccact
     1081 aaaaagatcg acaaattggc ctttgaaggg aaatgccttg gcgatctgat aagcaagaag
     1141 gggcggaaca gcgttgccga tctggttgtt gatggccgtc ttgctaccgt agaagatgaa
     1201 atcgtcaggg aaagactgca agcgcgcggc ctcacgctgt gacagagtgc gatcctgttc
     1261 ataatggatg tggcacccat tacctggacg gttgaagtac gtgttgatag tgtagcttgg
     1321 catatctggt aacaggcggc catagtaagt tgagcgtgat ccttttccct cagcataaga
     1381 cttacgaatt tgctccaaac gcttagacgg cacccattcg gggatgtctt tccaatttcc
     1441 tccgggcggc acgtgtttaa tgataagcat atggctgccg cgcggcacca ggccgctgct
     1501 gtgatgatga tgatgatggc tgctgcccat ggtatatctc cttcttaaag ttaaacaaaa
     1561 ttatttctag aggggaattg ttatccgctc acaattcccc tatagtgagt cgtattaatt
     1621 tcgcgggatc gagatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc
     1681 cacaggtgcg gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg
     1741 ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg
     1801 gggactgttg ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg
     1861 cctcaaccta ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga
     1921 tcccggacac catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag
     1981 agagtcaatt cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg
     2041 ccggtgtctc ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga
     2101 aaacgcggga aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg
     2161 cacaacaact ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc
     2221 tgcacgcgcc gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca
     2281 gcgtggtggt gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca
     2341 atcttctcgc gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg
     2401 ccattgctgt ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc
     2461 agacacccat caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc
     2521 atctggtcgc attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct
     2581 cggcgcgtct gcgtctggct ggctggcata aatatctcac tcgcaatcaa attcagccga
     2641 tagcggaacg ggaaggcgac tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc
     2701 tgaatgaggg catcgttccc actgcgatgc tggttgccaa cgatcagatg gcgctgggcg
     2761 caatgcgcgc cattaccgag tccgggctgc gcgttggtgc ggatatctcg gtagtgggat
     2821 acgacgatac cgaagacagc tcatgttata tcccgccgtt aaccaccatc aaacaggatt
     2881 ttcgcctgct ggggcaaacc agcgtggacc gcttgctgca actctctcag ggccaggcgg
     2941 tgaagggcaa tcagctgttg cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca
     3001 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
     3061 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtaagtta gctcactcat
     3121 taggcaccgg gatctcgacc gatgcccttg agagccttca acccagtcag ctccttccgg
     3181 tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc
     3241 gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc
     3301 gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc
     3361 ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg
     3421 gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc
     3481 cccattatga ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca ggccatgctg
     3541 tccaggcagg tagatgacga ccatcaggga cagcttcaag gatcgctcgc ggctcttacc
     3601 agcctaactt cgatcattgg accgctgatc gtcacggcga tttatgccgc ctcggcgagc
     3661 acatggaacg ggttggcatg gattgtaggc gccgccctat accttgtctg cctccccgcg
     3721 ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg cggcacctcg
     3781 ctaacggatt caccactcca agaattggag ccaatcaatt cttgcggaga actgtgaatg
     3841 cgcaaaccaa cccttggcag aacatatcca tcgcgtccgc catctccagc agccgcacgc
     3901 ggcgcatctc gggcagcgtt gggtcctggc cacgggtgcg catgatcgtg ctcctgtcgt
     3961 tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa tcaccgatac
     4021 gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca acaacatgaa
     4081 tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg ccctgcacca
     4141 ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg
     4201 tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata
     4261 ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc
     4321 gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaatcccc
     4381 cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta
     4441 tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg
     4501 cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc
     4561 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     4621 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     4681 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa
     4741 ctatgcggca tcagagcaga ttgtactgag agtgcaccat tgcggtgtga aataccgcac
     4801 agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg
     4861 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4921 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4981 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     5041 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     5101 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     5161 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     5221 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     5281 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     5341 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     5401 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
     5461 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5521 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5581 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5641 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5701 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5761 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5821 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5881 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5941 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     6001 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     6061 aatagtttgc gcaacgttgt tgccattgct gcaggcatcg tggtgtcacg ctcgtcgttt
     6121 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     6181 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     6241 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     6301 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     6361 cggcgaccga gttgctcttg cccggcgtca acacgggata ataccgcgcc acatagcaga
     6421 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6481 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6541 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6601 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6661 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6721 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     6781 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtcttcaa
     6841 aaa
//
