LOCUS       sequence-437745-        6885 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 6885)
  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 6885)
  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: POC1467
FEATURES             Location/Qualifiers
     source          1..6885
                     /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(1516..1533)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1543..1560)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1579..1601
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1616..1640
                     /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(1640..1659)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1641..1659)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1743..1762)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1968..2045
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2046..3128
                     /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(2065..2084)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4440..4631
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4625..4647)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4733..4872
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4788..4807
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4887..4904)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5058..5646)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5138..5157)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5817..6608,6609..6677))
                     /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     6440..6459
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6678..6782)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6850..6868
                     /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 gtcaagttca cttaacttgg aagatacgtg
     1501 gttgggaatc atatggctgc cgcgcggcac caggccgctg ctgtgatgat gatgatgatg
     1561 gctgctgccc atggtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
     1621 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc
     1681 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     1741 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     1801 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
     1861 ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
     1921 ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat
     1981 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     2041 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     2101 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     2161 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     2221 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     2281 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     2341 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     2401 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     2461 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     2521 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     2581 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     2641 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     2701 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     2761 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     2821 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     2881 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     2941 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     3001 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     3061 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     3121 ggcagtgagc gcaacgcaat taatgtaagt tagctcactc attaggcacc gggatctcga
     3181 ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg gggcatgact
     3241 atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca ggtgccggca
     3301 gcgctctggg tcattttcgg cgaggaccgc tttcgctgga gcgcgacgat gatcggcctg
     3361 tcgcttgcgg tattcggaat cttgcacgcc ctcgctcaag ccttcgtcac tggtcccgcc
     3421 accaaacgtt tcggcgagaa gcaggccatt atcgccggca tggcggccga cgcgctgggc
     3481 tacgtcttgc tggcgttcgc gacgcgaggc tggatggcct tccccattat gattcttctc
     3541 gcttccggcg gcatcgggat gcccgcgttg caggccatgc tgtccaggca ggtagatgac
     3601 gaccatcagg gacagcttca aggatcgctc gcggctctta ccagcctaac ttcgatcatt
     3661 ggaccgctga tcgtcacggc gatttatgcc gcctcggcga gcacatggaa cgggttggca
     3721 tggattgtag gcgccgccct ataccttgtc tgcctccccg cgttgcgtcg cggtgcatgg
     3781 agccgggcca cctcgacctg aatggaagcc ggcggcacct cgctaacgga ttcaccactc
     3841 caagaattgg agccaatcaa ttcttgcgga gaactgtgaa tgcgcaaacc aacccttggc
     3901 agaacatatc catcgcgtcc gccatctcca gcagccgcac gcggcgcatc tcgggcagcg
     3961 ttgggtcctg gccacgggtg cgcatgatcg tgctcctgtc gttgaggacc cggctaggct
     4021 ggcggggttg ccttactggt tagcagaatg aatcaccgat acgcgagcga acgtgaagcg
     4081 actgctgctg caaaacgtct gcgacctgag caacaacatg aatggtcttc ggtttccgtg
     4141 tttcgtaaag tctggaaacg cggaagtcag cgccctgcac cattatgttc cggatctgca
     4201 tcgcaggatg ctgctggcta ccctgtggaa cacctacatc tgtattaacg aagcgctggc
     4261 attgaccctg agtgattttt ctctggtccc gccgcatcca taccgccagt tgtttaccct
     4321 cacaacgttc cagtaaccgg gcatgttcat catcagtaac ccgtatcgtg agcatcctct
     4381 ctcgtttcat cggtatcatt acccccatga acagaaatcc cccttacacg gaggcatcag
     4441 tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa
     4501 cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc
     4561 ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg
     4621 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg
     4681 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca
     4741 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca
     4801 gattgtactg agagtgcacc attgcggtgt gaaataccgc acagatgcgt aaggagaaaa
     4861 taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4921 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4981 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5041 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5101 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5161 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5221 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5281 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5341 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     5401 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     5461 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     5521 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     5581 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     5641 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     5701 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     5761 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     5821 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     5881 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5941 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6001 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6061 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6121 gttgccattg ctgcaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6181 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6241 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6301 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6361 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     6421 tgcccggcgt caacacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     6481 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     6541 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     6601 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     6661 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     6721 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     6781 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta
     6841 acctataaaa ataggcgtat cacgaggccc tttcgtcttc aaaaa
//
