LOCUS       sequence-437786-        6867 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 6867)
  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 6867)
  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: POC1465
FEATURES             Location/Qualifiers
     source          1..6867
                     /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(1498..1515)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1525..1542)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1561..1583
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1598..1622
                     /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(1622..1641)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1623..1641)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1725..1744)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1950..2027
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2028..3110
                     /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(2047..2066)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4422..4613
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4607..4629)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4715..4854
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4770..4789
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4869..4886)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5040..5628)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5120..5139)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5799..6590,6591..6659))
                     /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     6422..6441
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6660..6764)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6832..6850
                     /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 tcatatggct
     1501 gccgcgcggc accaggccgc tgctgtgatg atgatgatga tggctgctgc ccatggtata
     1561 tctccttctt aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt
     1621 cccctatagt gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct acgccggacg
     1681 catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata tcgccgacat
     1741 caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt tcggcgtggg
     1801 tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc atgcaccatt
     1861 ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc taatgcagga
     1921 gtcgcataag ggagagcgtc gagatcccgg acaccatcga atggcgcaaa acctttcgcg
     1981 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa
     2041 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga
     2101 accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc
     2161 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg
     2221 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat
     2281 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg
     2341 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta
     2401 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg
     2461 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag
     2521 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt
     2581 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc
     2641 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg
     2701 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg
     2761 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg
     2821 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc
     2881 cgttaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc
     2941 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga
     3001 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     3061 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     3121 attaatgtaa gttagctcac tcattaggca ccgggatctc gaccgatgcc cttgagagcc
     3181 ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg
     3241 actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcattttc
     3301 ggcgaggacc gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga
     3361 atcttgcacg ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag
     3421 aagcaggcca ttatcgccgg catggcggcc gacgcgctgg gctacgtctt gctggcgttc
     3481 gcgacgcgag gctggatggc cttccccatt atgattcttc tcgcttccgg cggcatcggg
     3541 atgcccgcgt tgcaggccat gctgtccagg caggtagatg acgaccatca gggacagctt
     3601 caaggatcgc tcgcggctct taccagccta acttcgatca ttggaccgct gatcgtcacg
     3661 gcgatttatg ccgcctcggc gagcacatgg aacgggttgg catggattgt aggcgccgcc
     3721 ctataccttg tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc
     3781 tgaatggaag ccggcggcac ctcgctaacg gattcaccac tccaagaatt ggagccaatc
     3841 aattcttgcg gagaactgtg aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt
     3901 ccgccatctc cagcagccgc acgcggcgca tctcgggcag cgttgggtcc tggccacggg
     3961 tgcgcatgat cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg
     4021 gttagcagaa tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt
     4081 ctgcgacctg agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa
     4141 cgcggaagtc agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc
     4201 taccctgtgg aacacctaca tctgtattaa cgaagcgctg gcattgaccc tgagtgattt
     4261 ttctctggtc ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc
     4321 gggcatgttc atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca
     4381 ttacccccat gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa
     4441 ccgcccttaa catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca
     4501 acgagctgga cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg
     4561 agctttaccg cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     4621 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     4681 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     4741 atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     4801 ccattgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgctct
     4861 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     4921 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     4981 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     5041 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     5101 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     5161 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     5221 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     5281 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     5341 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     5401 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     5461 aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc
     5521 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     5581 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     5641 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     5701 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     5761 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     5821 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     5881 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     5941 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     6001 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     6061 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctgcaggc
     6121 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     6181 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     6241 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     6301 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     6361 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaacacgg
     6421 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     6481 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     6541 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     6601 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     6661 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     6721 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     6781 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
     6841 atcacgaggc cctttcgtct tcaaaaa
//
