LOCUS       sequence-437744-        6888 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 6888)
  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 6888)
  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: POC1463
FEATURES             Location/Qualifiers
     source          1..6888
                     /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(1519..1536)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1546..1563)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1582..1604
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1619..1643
                     /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(1643..1662)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1644..1662)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1746..1765)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1971..2048
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2049..3131
                     /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(2068..2087)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4443..4634
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4628..4650)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4736..4875
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4791..4810
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4890..4907)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5061..5649)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5141..5160)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5820..6611,6612..6680))
                     /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     6443..6462
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6681..6785)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6853..6871
                     /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 atccttaaag cgacggctta atgttgcgtt cataatactc
      361 tgagggtgta atgtttccac gcataaacaa ctggtaatca ttttccgggt ggtggatata
      421 gtatttacca gatccatcct cgccgtgttg taatggggga aggtcagata acgcatcttt
      481 tactgacacc caagactctt tatcttccgt cagcttctga ggtggttcga tcgagaacag
      541 cggatcattg ctgcccacaa gaattacacg cttacggcgt tgtggaattg cgtaatgttc
      601 cgaatttaaa atccaaccat tcatggtttt cattgtggac gagaattcct ccttaacacg
      661 ttcaaagacc tttccttttt ccatgttcaa caggccggcg acgttctcga aaatgaatcc
      721 attcggctta atctcgttaa gcagcgactt gtagtgcata aacagactgt tgcgtgggtc
      781 gtccattgta cgcgcgttgc cagccgtgct aaatccctgg cacggaggcc cgcccagaat
      841 ccacagctgt ttatcaaaat ataatttctt aaagccagaa atcttgtctg cgatcttaga
      901 aaaagtttcg ttgtccgaaa tgcttccgca caaaacttct ttgtgtacgt tgttcgagta
      961 tgtctgtaag tacttttcct caatgtcatt agctaacaat ggagtccacc cggcccactt
     1021 aaagcccaac cccaatccgc ctgcaccact gaataagtca atgtagtagc cggtattacc
     1081 gatcgctttt tcaatttctt ttgcaataag aaacgcaaga aagggaggca cggcattacc
     1141 gatttgcgta ttaatcgccg tttgacctcc aaaaaagata aagtcatcag ggaacgactg
     1201 cagacgtgca gcttcgcgtt gtgaaagtac gcgatcttgc tcgtagtgaa tgtggcatcc
     1261 gttgccagga cgattgaaat aagtattgat cgtataagct ggcatatcgg gcaataaacg
     1321 cccgtagtat gtgctgcgcg acccttttcc ttgagcatag ctgtcacgaa tctgttcgat
     1381 acgtttcgat ggtacatcca agggaatatt cttccagttt cccccaggcg ggacagagtt
     1441 aacgatcatc atatccagct cacttaattt ggcggccttg tggtttggaa tcatgaatgt
     1501 ttcagacttg gtcatatggc tgccgcgcgg caccaggccg ctgctgtgat gatgatgatg
     1561 atggctgctg cccatggtat atctccttct taaagttaaa caaaattatt tctagagggg
     1621 aattgttatc cgctcacaat tcccctatag tgagtcgtat taatttcgcg ggatcgagat
     1681 ctcgatcctc tacgccggac gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc
     1741 tggcgcctat atcgccgaca tcaccgatgg ggaagatcgg gctcgccact tcgggctcat
     1801 gagcgcttgt ttcggcgtgg gtatggtggc aggccccgtg gccgggggac tgttgggcgc
     1861 catctccttg catgcaccat tccttgcggc ggcggtgctc aacggcctca acctactact
     1921 gggctgcttc ctaatgcagg agtcgcataa gggagagcgt cgagatcccg gacaccatcg
     1981 aatggcgcaa aacctttcgc ggtatggcat gatagcgccc ggaagagagt caattcaggg
     2041 tggtgaatgt gaaaccagta acgttatacg atgtcgcaga gtatgccggt gtctcttatc
     2101 agaccgtttc ccgcgtggtg aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag
     2161 tggaagcggc gatggcggag ctgaattaca ttcccaaccg cgtggcacaa caactggcgg
     2221 gcaaacagtc gttgctgatt ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc
     2281 aaattgtcgc ggcgattaaa tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga
     2341 tggtagaacg aagcggcgtc gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac
     2401 gcgtcagtgg gctgatcatt aactatccgc tggatgacca ggatgccatt gctgtggaag
     2461 ctgcctgcac taatgttccg gcgttatttc ttgatgtctc tgaccagaca cccatcaaca
     2521 gtattatttt ctcccatgaa gacggtacgc gactgggcgt ggagcatctg gtcgcattgg
     2581 gtcaccagca aatcgcgctg ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc
     2641 tggctggctg gcataaatat ctcactcgca atcaaattca gccgatagcg gaacgggaag
     2701 gcgactggag tgccatgtcc ggttttcaac aaaccatgca aatgctgaat gagggcatcg
     2761 ttcccactgc gatgctggtt gccaacgatc agatggcgct gggcgcaatg cgcgccatta
     2821 ccgagtccgg gctgcgcgtt ggtgcggata tctcggtagt gggatacgac gataccgaag
     2881 acagctcatg ttatatcccg ccgttaacca ccatcaaaca ggattttcgc ctgctggggc
     2941 aaaccagcgt ggaccgcttg ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc
     3001 tgttgcccgt ctcactggtg aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct
     3061 ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa
     3121 gcgggcagtg agcgcaacgc aattaatgta agttagctca ctcattaggc accgggatct
     3181 cgaccgatgc ccttgagagc cttcaaccca gtcagctcct tccggtgggc gcggggcatg
     3241 actatcgtcg ccgcacttat gactgtcttc tttatcatgc aactcgtagg acaggtgccg
     3301 gcagcgctct gggtcatttt cggcgaggac cgctttcgct ggagcgcgac gatgatcggc
     3361 ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc aagccttcgt cactggtccc
     3421 gccaccaaac gtttcggcga gaagcaggcc attatcgccg gcatggcggc cgacgcgctg
     3481 ggctacgtct tgctggcgtt cgcgacgcga ggctggatgg ccttccccat tatgattctt
     3541 ctcgcttccg gcggcatcgg gatgcccgcg ttgcaggcca tgctgtccag gcaggtagat
     3601 gacgaccatc agggacagct tcaaggatcg ctcgcggctc ttaccagcct aacttcgatc
     3661 attggaccgc tgatcgtcac ggcgatttat gccgcctcgg cgagcacatg gaacgggttg
     3721 gcatggattg taggcgccgc cctatacctt gtctgcctcc ccgcgttgcg tcgcggtgca
     3781 tggagccggg ccacctcgac ctgaatggaa gccggcggca cctcgctaac ggattcacca
     3841 ctccaagaat tggagccaat caattcttgc ggagaactgt gaatgcgcaa accaaccctt
     3901 ggcagaacat atccatcgcg tccgccatct ccagcagccg cacgcggcgc atctcgggca
     3961 gcgttgggtc ctggccacgg gtgcgcatga tcgtgctcct gtcgttgagg acccggctag
     4021 gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag cgaacgtgaa
     4081 gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc ttcggtttcc
     4141 gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg ttccggatct
     4201 gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta acgaagcgct
     4261 ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc agttgtttac
     4321 cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc gtgagcatcc
     4381 tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac acggaggcat
     4441 cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga agccagacat
     4501 taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac atctgtgaat
     4561 cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc ggtgatgacg
     4621 gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg
     4681 ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag
     4741 ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg cggcatcaga
     4801 gcagattgta ctgagagtgc accattgcgg tgtgaaatac cgcacagatg cgtaaggaga
     4861 aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     4921 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4981 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5041 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5101 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5161 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5221 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5281 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5341 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5401 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5461 gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc
     5521 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5581 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5641 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5701 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5761 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5821 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     5881 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     5941 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6001 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6061 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6121 gttgttgcca ttgctgcagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6181 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6241 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6301 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6361 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6421 tcttgcccgg cgtcaacacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6481 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6541 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6601 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6661 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6721 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6781 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     6841 ttaacctata aaaataggcg tatcacgagg ccctttcgtc ttcaaaaa
//
