LOCUS       sequence-439154-        6434 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 6434)
  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 6434)
  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: POC1468
FEATURES             Location/Qualifiers
     source          1..6434
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1371..1393
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1408..1432
                     /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(1432..1451)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1433..1451)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1539..1558)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1764..1841
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1842..2924
                     /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(1861..1880)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3733..3924
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3918..3940)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4026..4165
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4081..4100
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4180..4197)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4351..4939)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4431..4450)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5050..5069)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5061..5876
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5061..5876
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5134..5153)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(5968..6423)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6105..6126)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6317..6336
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttaa
      181 tgatagctaa ggaggacagc atctcgatat actgctgggc tgtgatcttc ttgcgcatga
      241 atgactgata aatattttgt ggctcacaaa cgtagttctt ccaagagccg tcctcattag
      301 gcgacaaaag gggtaagtct gaaagggcct cctttacgga aatggcactg cttaaggcgt
      361 caaacagggt cttttctcct ttaagagacg taattgggat agggggctcg ctgttcttcg
      421 ttccggtgga gtccccgatg atgactacgc ggttgcggcg ttggggcaca ccgtaatcca
      481 ccgaattaag tttatatacg aaaagattcg acaccgtctt agacaattcc gatttaacca
      541 tctcaaagaa tgctcccttc tccatcgaca acaggccagt cacgttttca aaaataaaac
      601 cgtctggctt gacttctttc aggatactca cgtaactttc gaacagccag ttgcgctcgt
      661 ccacgattga acgttttttt cctgcagtcg aaaacccttg gcagggtggt cccccaagaa
      721 caaacaatgg acggtccggg ttcgacttct ttgcgatcag gcacttttga atgatagtct
      781 cactgacctt ttcgtcgcgg atatccccaa gcactacttc attgtgaata ttattcatgt
      841 aggtggtcag cgcccattta tcgatgtcat ttgcgatgat cggtttccac ccggcccaaa
      901 ggaaacctaa ggaaaggccc cccaccccac taaaaagatc gacaaattgg cctttgaagg
      961 gaaatgcctt ggcgatctga taagcaagaa ggggcggaac agcgttgccg atctggttgt
     1021 tgatggccgt cttgctaccg tagaagatga aatcgtcagg gaaagactgc aagcgcgcgg
     1081 cctcacgctg tgacagagtg cgatcctgtt cataatggat gtggcaccca ttacctggac
     1141 ggttgaagta cgtgttgata gtgtagcttg gcatatctgg taacaggcgg ccatagtaag
     1201 ttgagcgtga tccttttccc tcagcataag acttacgaat ttgctccaaa cgcttagacg
     1261 gcacccattc ggggatgtct ttccaatttc ctccgggcgg cacgtgttta atgataagca
     1321 tgtcaagttc acttaacttg gaagatacgt ggttgggaat catatgtata tctccttctt
     1381 aaagttaaac aaaattattt ctagagggga attgttatcc gctcacaatt cccctatagt
     1441 gagtcgtatt aatttcgcgg gatcgagatc gatctcgatc ctctacgccg gacgcatcgt
     1501 ggccggcatc accggcgcca caggtgcggt tgctggcgcc tatatcgccg acatcaccga
     1561 tggggaagat cgggctcgcc acttcgggct catgagcgct tgtttcggcg tgggtatggt
     1621 ggcaggcccc gtggccgggg gactgttggg cgccatctcc ttgcatgcac cattccttgc
     1681 ggcggcggtg ctcaacggcc tcaacctact actgggctgc ttcctaatgc aggagtcgca
     1741 taagggagag cgtcgagatc ccggacacca tcgaatggcg caaaaccttt cgcggtatgg
     1801 catgatagcg cccggaagag agtcaattca gggtggtgaa tgtgaaacca gtaacgttat
     1861 acgatgtcgc agagtatgcc ggtgtctctt atcagaccgt ttcccgcgtg gtgaaccagg
     1921 ccagccacgt ttctgcgaaa acgcgggaaa aagtggaagc ggcgatggcg gagctgaatt
     1981 acattcccaa ccgcgtggca caacaactgg cgggcaaaca gtcgttgctg attggcgttg
     2041 ccacctccag tctggccctg cacgcgccgt cgcaaattgt cgcggcgatt aaatctcgcg
     2101 ccgatcaact gggtgccagc gtggtggtgt cgatggtaga acgaagcggc gtcgaagcct
     2161 gtaaagcggc ggtgcacaat cttctcgcgc aacgcgtcag tgggctgatc attaactatc
     2221 cgctggatga ccaggatgcc attgctgtgg aagctgcctg cactaatgtt ccggcgttat
     2281 ttcttgatgt ctctgaccag acacccatca acagtattat tttctcccat gaagacggta
     2341 cgcgactggg cgtggagcat ctggtcgcat tgggtcacca gcaaatcgcg ctgttagcgg
     2401 gcccattaag ttctgtctcg gcgcgtctgc gtctggctgg ctggcataaa tatctcactc
     2461 gcaatcaaat tcagccgata gcggaacggg aaggcgactg gagtgccatg tccggttttc
     2521 aacaaaccat gcaaatgctg aatgagggca tcgttcccac tgcgatgctg gttgccaacg
     2581 atcagatggc gctgggcgca atgcgcgcca ttaccgagtc cgggctgcgc gttggtgcgg
     2641 acatctcggt agtgggatac gacgataccg aagacagctc atgttatatc ccgccgttaa
     2701 ccaccatcaa acaggatttt cgcctgctgg ggcaaaccag cgtggaccgc ttgctgcaac
     2761 tctctcaggg ccaggcggtg aagggcaatc agctgttgcc cgtctcactg gtgaaaagaa
     2821 aaaccaccct ggcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa
     2881 tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
     2941 gtaagttagc tcactcatta ggcaccggga tctcgaccga tgcccttgag agccttcaac
     3001 ccagtcagct ccttccggtg ggcgcggggc atgactatcg tcgccgcact tatgactgtc
     3061 ttctttatca tgcaactcgt aggacaggtg ccggcagcgc tctgggtcat tttcggcgag
     3121 gaccgctttc gctggagcgc gacgatgatc ggcctgtcgc ttgcggtatt cggaatcttg
     3181 cacgccctcg ctcaagcctt cgtcactggt cccgccacca aacgtttcgg cgagaagcag
     3241 gccattatcg ccggcatggc ggccccacgg gtgcgcatga tcgtgctcct gtcgttgagg
     3301 acccggctag gctggcgggg ttgccttact ggttagcaga atgaatcacc gatacgcgag
     3361 cgaacgtgaa gcgactgctg ctgcaaaacg tctgcgacct gagcaacaac atgaatggtc
     3421 ttcggtttcc gtgtttcgta aagtctggaa acgcggaagt cagcgccctg caccattatg
     3481 ttccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta
     3541 acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc
     3601 agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc
     3661 gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa tcccccttac
     3721 acggaggcat cagtgaccaa acaggaaaaa accgccctta acatggcccg ctttatcaga
     3781 agccagacat taacgcttct ggagaaactc aacgagctgg acgcggatga acaggcagac
     3841 atctgtgaat cgcttcacga ccacgctgat gagctttacc gcagctgcct cgcgcgtttc
     3901 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     3961 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     4021 cggggcgcag ccatgaccca gtcacgtagc gatagcggag tgtatactgg cttaactatg
     4081 cggcatcaga gcagattgta ctgagagtgc accattgcgg tgtgaaatac cgcacagatg
     4141 cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4201 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4261 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4321 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4381 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4441 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4501 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4561 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4621 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4681 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4741 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt
     4801 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4861 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4921 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4981 gaacgaaaac tcacgttaag ggattttggt catgaacaat aaaactgtct gcttacataa
     5041 acagtaatac aaggggtgtt atgagccata ttcaacggga aacgtcttgc tctaggccgc
     5101 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     5161 ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca gagttgtttc
     5221 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     5281 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
     5341 catggttact caccactgcg atccccggga aaacagcatt ccaggtatta gaagaatatc
     5401 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
     5461 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat
     5521 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     5581 ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg
     5641 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     5701 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     5761 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     5821 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaagaat
     5881 taattcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5941 cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg
     6001 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     6061 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     6121 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     6181 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     6241 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     6301 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     6361 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     6421 cgtcccattc gcca
//
