LOCUS       sequence-437773-        6764 bp    DNA     circular SYN 15-APR-2025
DEFINITION  BsaI-associated switch methylase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6764)
  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 6764)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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: POC1471
FEATURES             Location/Qualifiers
     source          1..6764
                     /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(1396..1413)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1423..1440)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1459..1481
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1496..1520
                     /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(1520..1539)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1521..1539)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1623..1642)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1848..1925
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     complement(1945..1964)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4319..4510
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4504..4526)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4612..4751
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4667..4686
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4766..4783)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4937..5525)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5017..5036)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5696..6487,6488..6556))
                     /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     6319..6338
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6557..6661)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6729..6747
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ttctcatgtt 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 atccttattc cccttcttta aactcgtaaa caccgttcga
      361 ttcaaaatag gcatttgcct cattcaggcg ggccgctgct aacttgtaaa atgtgtcgtc
      421 aatttctgca gcgtaactgc ggcgaccaag taatgacgat gccacactag ccgagcacag
      481 acctccaaat ggttcccaga ctgtatcgcc gacattactg gctgctgcta cctgagtaag
      541 catcaggtcc aggggctttt ggttcagatg tgctgctgac tgtttggtgg gtttatacgt
      601 gcgaggtgcg gagcgttcca ttgtcccttt caaacgctca ctatcggcca acggcggacg
      661 gctccacacg tttgtgactc cattgcggtg attccatgta gtgcgtaaac tatcccaatc
      721 atgagctgaa atcatagttt tgccatccag actgaaatag ggccaagaag tttttttacc
      781 atattgcatg cagtactccg ccatcttttg gaccgcgtca ccaggcggcc agtaccacag
      841 ccagtctgct gtcaaatatt tacgggtcgc agcgttcttc acaccacacg cttcattcga
      901 ctttgaaagg gacaggcccg agcgacgcca ctctgcgcgc aaccatgact ttgcgtttaa
      961 tgtaagtccg tctccagttt ccaggtacac cgtacggcga taaagggccg ccacctctgt
     1021 taccacgggc aattgacgga ttgttttccc gttaacattg ccggcaatgt gcgccagtcc
     1081 tttatcccag acagctaact gcacgtattc ccatccggtg gacaataata aagggtggac
     1141 cgtggcccag cctacttctg tgttccaaaa ccacaaagaa gtggaaggct tggccgcttt
     1201 ggcccacgcc agcacgtgtg gcttatacca gtccgtcaac ccggcagcgt caacggtgtc
     1261 cccgcgaaat ccacgaacac cgtatgcccc atccgataca attaagtccg gactaggcca
     1321 gcgcccgtag acttccgata cgttaccctt ataaaggtgg gaaagcttaa ggtttgtgtc
     1381 cttctttgcc atatggctgc cgcgcggcac caggccgctg ctgtgatgat gatgatgatg
     1441 gctgctgccc atggtatatc tccttcttaa agttaaacaa aattatttct agaggggaat
     1501 tgttatccgc tcacaattcc cctatagtga gtcgtattaa tttcgcggga tcgagatctc
     1561 gatcctctac gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg
     1621 cgcctatatc gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag
     1681 cgcttgtttc ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat
     1741 ctccttgcat gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg
     1801 ctgcttccta atgcaggagt cgcataaggg agagcgtcga gatcccggac accatcgaat
     1861 ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga agagagtcaa ttcagggtgg
     1921 tgaatgtgaa accagtaacg ttatacgatg tcgcagagta tgccggtgtc tcttatcaga
     1981 ccgtttcccg cgtggtgaac caggccagcc acgtttctgc gaaaacgcgg gaaaaagtgg
     2041 aagcggcgat ggcggagctg aattacattc ccaaccgcgt ggcacaacaa ctggcgggca
     2101 aacagtcgtt gctgattggc gttgccacct ccagtctggc cctgcacgcg ccgtcgcaaa
     2161 ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc cagcgtggtg gtgtcgatgg
     2221 tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca caatcttctc gcgcaacgcg
     2281 tcagtgggct gatcattaac tatccgctgg atgaccagga tgccattgct gtggaagctg
     2341 cctgcactaa tgttccggcg ttatttcttg atgtctctga ccagacaccc atcaacagta
     2401 ttattttctc ccatgaagac ggtacgcgac tgggcgtgga gcatctggtc gcattgggtc
     2461 accagcaaat cgcgctgtta gcgggcccat taagttctgt ctcggcgcgt ctgcgtctgg
     2521 ctggctggca taaatatctc actcgcaatc aaattcagcc gatagcggaa cgggaaggcg
     2581 actggagtgc catgtccggt tttcaacaaa ccatgcaaat gctgaatgag ggcatcgttc
     2641 ccactgcgat gctggttgcc aacgatcaga tggcgctggg cgcaatgcgc gccattaccg
     2701 agtccgggct gcgcgttggt gcggatatct cggtagtggg atacgacgat accgaagaca
     2761 gctcatgtta tatcccgccg ttaaccacca tcaaacagga ttttcgcctg ctggggcaaa
     2821 ccagcgtgga ccgcttgctg caactctctc agggccaggc ggtgaagggc aatcagctgt
     2881 tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc caatacgcaa accgcctctc
     2941 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca gtttcccgac tggaaagcgg
     3001 gcagtgagcg caacgcaatt aatgtaagtt agctcactca ttaggcaccg ggatctcgac
     3061 cgatgccctt gagagccttc aacccagtca gctccttccg gtgggcgcgg ggcatgacta
     3121 tcgtcgccgc acttatgact gtcttcttta tcatgcaact cgtaggacag gtgccggcag
     3181 cgctctgggt cattttcggc gaggaccgct ttcgctggag cgcgacgatg atcggcctgt
     3241 cgcttgcggt attcggaatc ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca
     3301 ccaaacgttt cggcgagaag caggccatta tcgccggcat ggcggccgac gcgctgggct
     3361 acgtcttgct ggcgttcgcg acgcgaggct ggatggcctt ccccattatg attcttctcg
     3421 cttccggcgg catcgggatg cccgcgttgc aggccatgct gtccaggcag gtagatgacg
     3481 accatcaggg acagcttcaa ggatcgctcg cggctcttac cagcctaact tcgatcattg
     3541 gaccgctgat cgtcacggcg atttatgccg cctcggcgag cacatggaac gggttggcat
     3601 ggattgtagg cgccgcccta taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga
     3661 gccgggccac ctcgacctga atggaagccg gcggcacctc gctaacggat tcaccactcc
     3721 aagaattgga gccaatcaat tcttgcggag aactgtgaat gcgcaaacca acccttggca
     3781 gaacatatcc atcgcgtccg ccatctccag cagccgcacg cggcgcatct cgggcagcgt
     3841 tgggtcctgg ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg
     3901 gcggggttgc cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga
     3961 ctgctgctgc aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt
     4021 ttcgtaaagt ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat
     4081 cgcaggatgc tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca
     4141 ttgaccctga gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc
     4201 acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc
     4261 tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt
     4321 gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac
     4381 gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct
     4441 tcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga
     4501 aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg
     4561 gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat
     4621 gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag
     4681 attgtactga gagtgcacca ttgcggtgtg aaataccgca cagatgcgta aggagaaaat
     4741 accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4801 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4861 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4921 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4981 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5041 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5101 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5161 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5221 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5281 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5341 tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
     5401 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5461 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5521 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     5581 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5641 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5701 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5761 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     5821 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     5881 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     5941 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6001 ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6061 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6121 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6181 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6241 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6301 gcccggcgtc aacacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6361 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6421 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6481 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6541 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6601 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6661 gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa
     6721 cctataaaaa taggcgtatc acgaggccct ttcgtcttca agaa
//
