LOCUS       sequence-479109-        7919 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NovaIscB E. coli expression for recombinant purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7919)
  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 7919)
  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: "Nat 
            Biotechnol. 2025 May 7. doi: 10.1038/s41587-025-02655-3."
COMMENT     Sequence Label: pHS2096
FEATURES             Location/Qualifiers
     source          1..7919
                     /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      2743..2790
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2965..2970,2971..2987,2988..2993))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3020..3038)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3106..3210
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3211..3279,3280..4071)
                     /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(3429..3448)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4242..4830
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4731..4750
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4984..5001
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5016..5155
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5081..5100)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5241..5263
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5257..5448)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6760..7842)
                     /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     7804..7823
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(7843..7919,1))
                     /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 aagaacatca gactgcacgg ccctggcgga gagatcgtgg gcctgcaccc cgacctgaag
     1501 gaaacactca agagaaccat cctggaaggc cagcagggca atatcaacaa gctgttcagg
     1561 aaacttagaa aggtgtacaa gatcaccctg aaccagaaag actggcctgg caaaaacctg
     1621 accgatatcg ctaagaataa gctgccaggc cggctgccct tctgcaagga gcacttcgcc
     1681 gagaacgaga agttcaccac catcgagaag agcaccttcc ggctgacccc taccgccacc
     1741 cagctgctgc ggacccacat caacctgttt agaaagctga gcggcatcct gcccgtgacc
     1801 gacgtggccg tcgagctgaa caagttcgcc tttatgcagc tggacaaccc tgagatgaag
     1861 aagcgggaaa tcgacttctg ccacggccca ctgtgcggca ccggcggcct ggaagccgct
     1921 gttaaggagc agcaggatgg aaaatgcctg ctgtgtggaa aagaatctat cggccactac
     1981 caccacatcg tgcctaggtc ccgtagaggc agcaacacca tcgctaatat cgccggcctt
     2041 tgtcctaagt gccacgagct cgtgcacaag gacgccgata cagccgagtc tctgacagag
     2101 atgaaaaccg gcctgatgaa gaagtatgga ggcaccagcg tgctgaacca gatcatcccc
     2161 aagctggtgg aaaccctggc cgacctgttt cctggccact tccacgtgac aaacggctgg
     2221 aacaccaagg agttcagaga gaaacatcat ctggaaaagg atcacgacgt ggatgcttac
     2281 tgcatcgcct gcagccacct gaagcctgag gaaaccctgg tggaaactga gcctttcgag
     2341 atcctgcagt tcagaaagca caacagagcc atcattcacc accagaccga gcggacgtac
     2401 aagctggacg gcgtgacagt ggctaaaaat agaaagaaac ggatggaaca gaaaaccgac
     2461 agcctggaag attggtacgt ggacatggcc aaggaacacg gcaaaacaca ggccgatgcc
     2521 atgcggagca gactgaccgt gatcaagagc acacggtatt acaacacccc tggcagaatg
     2581 atgccaggca cagttttcct gtacgagggc aagagatacg tgatgaccgg ccagattaca
     2641 aacggcaagt actacagagc ctacggccag gagaagcgga acttccccgc tgtcaaggtg
     2701 aggattctga caaagaacac cggactagtc tttgtggcct agctagcata accccttggg
     2761 gcctctaaac gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatccc
     2821 gcaagaggcc cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg
     2881 tgccgaggat gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc
     2941 aatttaactg tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag
     3001 aattcttgaa gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata
     3061 ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     3121 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     3181 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     3241 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     3301 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     3361 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     3421 aaagttctgc tatgtggcgc ggtattatcc cgtgttgacg ccgggcaaga gcaactcggt
     3481 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     3541 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     3601 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     3661 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     3721 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     3781 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     3841 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     3901 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     3961 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     4021 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     4081 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     4141 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     4201 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     4261 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     4321 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     4381 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     4441 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     4501 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     4561 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     4621 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     4681 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     4741 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     4801 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     4861 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     4921 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     4981 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     5041 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     5101 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     5161 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     5221 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     5281 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     5341 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     5401 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     5461 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     5521 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     5581 ggcggtatgg atgcggcggg accagagaaa aatcactcag ggtcaatgcc agcgcttcgt
     5641 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     5701 cataatggtg cagggcgctg acttccgcgt ttacagactt tacgaaacac ggaaaccgaa
     5761 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     5821 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     5881 cctcaacgac aggagcacga tcatgcgcac ccgtggccag gacccaacgc tgcccgagat
     5941 gcgccgcgtg cggctgctgg agatggcgga cgcgatggat atgttctgcc aagggttggt
     6001 ttgcgcattc acagttctcc gcaagaattg attggctcca attcttggag tggtgaatcc
     6061 gttagcgagg tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga
     6121 cgcaacgcgg ggaggcagac aaggtatagg gcggcgccta caatccatgc caacccgttc
     6181 catgtgctcg ccgaggcggc ataaatcgcc gtgacgatca gcggtccaat gatcgaagtt
     6241 aggctggtaa gagccgcgag cgatccttga agctgtccct gatggtcgtc atctacctgc
     6301 ctggacagca tggcctgcaa cgcgggcatc ccgatgccgc cggaagcgag aagaatcata
     6361 atggggaagg ccatccagcc tcgcgtcgcg aacgccagca agacgtagcc cagcgcgtcg
     6421 gccgccatgc cggcgataat ggcctgcttc tcgccgaaac gtttggtggc gggaccagtg
     6481 acgaaggctt gagcgagggc gtgcaagatt ccgaataccg caagcgacag gccgatcatc
     6541 gtcgcgctcc agcgaaagcg gtcctcgccg aaaatgaccc agagcgctgc cggcacctgt
     6601 cctacgagtt gcatgataaa gaagacagtc ataagtgcgg cgacgatagt catgccccgc
     6661 gcccaccgga aggagctgac tgggttgaag gctctcaagg gcatcggtcg agatcccggt
     6721 gcctaatgag tgagctaact tacattaatt gcgttgcgct cactgcccgc tttccagtcg
     6781 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     6841 cgtattgggc gccagggtgg tttttctttt caccagtgag acgggcaaca gctgattgcc
     6901 cttcaccgcc tggccctgag agagttgcag caagcggtcc acgctggttt gccccagcag
     6961 gcgaaaatcc tgtttgatgg tggttaacgg cgggatataa catgagctgt cttcggtatc
     7021 gtcgtatccc actaccgaga tatccgcacc aacgcgcagc ccggactcgg taatggcgcg
     7081 cattgcgccc agcgccatct gatcgttggc aaccagcatc gcagtgggaa cgatgccctc
     7141 attcagcatt tgcatggttt gttgaaaacc ggacatggca ctccagtcgc cttcccgttc
     7201 cgctatcggc tgaatttgat tgcgagtgag atatttatgc cagccagcca gacgcagacg
     7261 cgccgagaca gaacttaatg ggcccgctaa cagcgcgatt tgctggtgac ccaatgcgac
     7321 cagatgctcc acgcccagtc gcgtaccgtc ttcatgggag aaaataatac tgttgatggg
     7381 tgtctggtca gagacatcaa gaaataacgc cggaacatta gtgcaggcag cttccacagc
     7441 aatggcatcc tggtcatcca gcggatagtt aatgatcagc ccactgacgc gttgcgcgag
     7501 aagattgtgc accgccgctt tacaggcttc gacgccgctt cgttctacca tcgacaccac
     7561 cacgctggca cccagttgat cggcgcgaga tttaatcgcc gcgacaattt gcgacggcgc
     7621 gtgcagggcc agactggagg tggcaacgcc aatcagcaac gactgtttgc ccgccagttg
     7681 ttgtgccacg cggttgggaa tgtaattcag ctccgccatc gccgcttcca ctttttcccg
     7741 cgttttcgca gaaacgtggc tggcctggtt caccacgcgg gaaacggtct gataagagac
     7801 accggcatac tctgcgacat cgtataacgt tactggtttc acattcacca ccctgaattg
     7861 actctcttcc gggcgctatc atgccatacc gcgaaaggtt ttgcgccatt cgatggtgt
//
