LOCUS       sequence-479361-        7871 bp    DNA     circular SYN 22-JUL-2025
DEFINITION  OrufIscB-REC E. coli expression for recombinant purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7871)
  AUTHORS   Kannan S, Altae-Tran H, Zhu S, Xu P, Strebinger D, Oshiro R, Faure 
            G, Moeller L, Pham J, Mears KS, Ni HM, Macrae RK, Zhang F
  TITLE     Evolution-guided protein design of IscB for persistent epigenome 
            editing in vivo.
  JOURNAL   Nat Biotechnol. 2025 May 7. doi: 10.1038/s41587-025-02655-3.
  PUBMED    40335752
REFERENCE   2  (bases 1 to 7871)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 22, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2025 May 7. doi: 10.1038/s41587-025-02655-3."
COMMENT     Sequence Label: pHS1625
FEATURES             Location/Qualifiers
     source          1..7871
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     207..226
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     310..329
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        310..328
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    329..353
                     /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)."
     RBS             368..390
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             484..501
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(514..537,538..573,574..597)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     terminator      2695..2742
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2917..2922,2923..2939,2940..2945))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2972..2990)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3058..3162
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3163..3231,3232..4023)
                     /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     complement(3381..3400)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4194..4782
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4683..4702
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4936..4953
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4968..5107
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5033..5052)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5193..5215
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5209..5400)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6712..7794)
                     /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     7756..7775
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(7795..7871,1))
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg
       61 ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac
      121 agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg
      181 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
      241 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
      301 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
      361 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcaccatcat
      421 tcaggccatc accataccgg acaccaccat cattcaggca gtcatcacca ttccagcagc
      481 ggcctggtgc cgcgcggcag ccatatggct agctggagcc atccgcagtt tgaaaaaggt
      541 ggtggtagcg gtggtggttc aggtggtagt gcatggtcac accctcagtt tgagaaagga
      601 tctgctgcgg gtggcgaaga agataagaaa ccggcaggtg gcgaaggtgg cggtgcccat
      661 atcaacctga aagtgaaagg tcaagacggc aacgaagtct ttttccgcat caaacgttct
      721 acccagctga aaaagctgat gaacgcatac tgtgaccgtc agtctgtaga catgaccgca
      781 attgctttcc tgtttgatgg tcgtcgcctg cgtgcggaac agaccccgga tgaactggag
      841 atggaagatg gcgacgaaat cgacgcaatg ctgcaccaga ctggtggctc aagtggatcc
      901 agatacgtgt acgtgctgga tgtggacggc aagcctctga tgcctacatg caggttcggc
      961 aaggtgcgga gaatgctgaa atctggccaa gctaaggccg tggacaccct gcctttcacc
     1021 atccagctga cctacaagcc cagaaccaga atcctgcaac ctgtgaccct gggccaggac
     1081 cccggcagaa ccaatatcgg aatggccgcc gtgcggttcg acggcaagga actgggaaga
     1141 ttccactgca tcacaagaaa caaggaaatc cctaagctga tggccgacag aatggcagcc
     1201 agaaaggcct cccggagagg cgagagactg gccagaaagc gcctggcccg gaaactgcac
     1261 accaccgcca agcacctgaa tggcagaatc ctgccaggat gtagcgagcc catcgccgtg
     1321 aaggacatca tcaacacaga gagcagattc aacaacagaa tcctgaccaa atgcaaagtg
     1381 tgcggcaaga acacccctct gagaagaaac gtgcgggaac tcctgctgga aaacatcgtg
     1441 cggtttctgc ctctggaaag cgagctgaag gaaacactca agagaaccat cctggaaggc
     1501 cagcagggca atatcaacaa gctgttcagg aaacttaaat tcaatcaaaa ggattggcct
     1561 ggcaaaaacc tgaccgatat cgctaagaat aagctgccag gccggctgcc cttctgcaag
     1621 gagcacttcg ccgagaacga gaagttcacc accatcgaga agagcacctt ccggctgacc
     1681 cctaccgcca cccagctgct gcggacccac atcaacctgt ttagaaagct gagcggcatc
     1741 ctgcccgtga ccgacgtggc cgtcgagctg aacaagttcg cctttatgca gctggacaac
     1801 cctgagatga agaagcggga aatcgacttc tgccacggcc cactgtgcgg caccggcggc
     1861 ctggaagccg ctgttaagga gcagcaggat ggaaaatgcc tgctgtgtgg aaaagaatct
     1921 atcggccact accaccacat cgtgcctagg tcccgtagag gcagcaacac catcgctaat
     1981 atcgccggcc tttgtcctaa gtgccacgag ctcgtgcaca aggacgccga tacagccgag
     2041 tctctgacag agatgaaaac cggcctgatg aagaagtatg gaggcaccag cgtgctgaac
     2101 cagatcatcc ccaagctggt ggaaaccctg gccgacctgt ttcctggcca cttccacgtg
     2161 acaaacggct ggaacaccaa ggagttcaga gagaaacatc atctggaaaa ggatcacgac
     2221 gtggatgctt actgcatcgc ctgcagccac ctgaagcctg aggaaaccct ggtggaaact
     2281 gagcctttcg agatcctgca gttcagaaag cacaacagag ccatcattca ccaccagacc
     2341 gagcggacgt acaagctgga cggcgtgaca gtggctaaaa atagaaagaa acggatggaa
     2401 cagaaaaccg acagcctgga agattggtac gtggacatgg ccaaggaaca cggcaaaaca
     2461 caggccgatg ccatgcggag cagactgacc gtgatcaaga gcacacggta ttacaacacc
     2521 cctggcagaa tgatgccagg cacagttttc ctgtacgagg gcaagagata cgtgatgacc
     2581 ggccagatta caaacggcaa gtactacaga gcctacggcc aggagaagcg gaacttcccc
     2641 gctgtcaagg tgaggattct gacaaagaac accggactag tctttgtggc ctagctagca
     2701 taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg aggaactata
     2761 tccggatatc ccgcaagagg cccggcagta ccggcataac caagcctatg cctacagcat
     2821 ccagggtgac ggtgccgagg atgacgatga gcgcattgtt agatttcata cacggtgcct
     2881 gactgcgtta gcaatttaac tgtgataaac taccgcatta aagcttatcg atgataagct
     2941 gtcaaacatg agaattcttg aagacgaaag ggcctcgtga tacgcctatt tttataggtt
     3001 aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc
     3061 ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa
     3121 taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc
     3181 cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa
     3241 acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa
     3301 ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg
     3361 atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgttga cgccgggcaa
     3421 gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc
     3481 acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc
     3541 atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta
     3601 accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag
     3661 ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgcagc aatggcaaca
     3721 acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca acaattaata
     3781 gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc
     3841 tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca
     3901 ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca
     3961 actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg
     4021 taactgtcag accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa
     4081 tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt
     4141 gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat
     4201 cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg
     4261 gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga
     4321 gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca cttcaagaac
     4381 tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt
     4441 ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag
     4501 cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc
     4561 gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag
     4621 gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca
     4681 gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt
     4741 cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc
     4801 tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc tgcgttatcc
     4861 cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc tcgccgcagc
     4921 cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgcct gatgcggtat
     4981 tttctcctta cgcatctgtg cggtatttca caccgcaatg gtgcactctc agtacaatct
     5041 gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat
     5101 ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc
     5161 ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc
     5221 accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag
     5281 cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt
     5341 taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac
     5401 tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga
     5461 gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga
     5521 gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg
     5581 ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat
     5641 gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttacagac tttacgaaac
     5701 acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc
     5761 gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca
     5821 gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtggcc aggacccaac
     5881 gctgcccgag atgcgccgcg tgcggctgct ggagatggcg gacgcgatgg atatgttctg
     5941 ccaagggttg gtttgcgcat tcacagttct ccgcaagaat tgattggctc caattcttgg
     6001 agtggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc
     6061 catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgcc tacaatccat
     6121 gccaacccgt tccatgtgct cgccgaggcg gcataaatcg ccgtgacgat cagcggtcca
     6181 atgatcgaag ttaggctggt aagagccgcg agcgatcctt gaagctgtcc ctgatggtcg
     6241 tcatctacct gcctggacag catggcctgc aacgcgggca tcccgatgcc gccggaagcg
     6301 agaagaatca taatggggaa ggccatccag cctcgcgtcg cgaacgccag caagacgtag
     6361 cccagcgcgt cggccgccat gccggcgata atggcctgct tctcgccgaa acgtttggtg
     6421 gcgggaccag tgacgaaggc ttgagcgagg gcgtgcaaga ttccgaatac cgcaagcgac
     6481 aggccgatca tcgtcgcgct ccagcgaaag cggtcctcgc cgaaaatgac ccagagcgct
     6541 gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc ggcgacgata
     6601 gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa gggcatcggt
     6661 cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg ctcactgccc
     6721 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     6781 agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa
     6841 cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt
     6901 ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct
     6961 gtcttcggta tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc
     7021 ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg
     7081 aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc
     7141 gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc
     7201 cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg
     7261 acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat
     7321 actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc
     7381 agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac
     7441 gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac
     7501 catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat
     7561 ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt
     7621 gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc
     7681 cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt
     7741 ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac
     7801 caccctgaat tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca
     7861 ttcgatggtg t
//
