LOCUS       sequence-479116-        7574 bp    DNA     circular SYN 21-JUL-2025
DEFINITION  WT OrufIscB E. coli expression for recombinant purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7574)
  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 7574)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 21, 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: pHS1623
FEATURES             Location/Qualifiers
     source          1..7574
                     /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      2398..2445
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2620..2625,2626..2642,2643..2648))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2675..2693)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2761..2865
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2866..2934,2935..3726)
                     /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(3084..3103)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3897..4485
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4386..4405
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4639..4656
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4671..4810
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4736..4755)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4896..4918
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4912..5103)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6415..7497)
                     /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     7459..7478
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(join(7498..7574,1))
                     /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 aacaacagac tgcggcctga gggatggctg
     1381 acccctaccg ccacccagct gctgcggacc cacatcaacc tgtttagaaa gctgagcggc
     1441 atcctgcccg tgaccgacgt ggccgtcgag ctgaacaagt tcgcctttat gcagctggac
     1501 aaccctgaga tgaagaagcg ggaaatcgac ttctgccacg gcccactgtg cggcaccggc
     1561 ggcctggaag ccgctgttaa ggagcagcag gatggaaaat gcctgctgtg tggaaaagaa
     1621 tctatcggcc actaccacca catcgtgcct aggtcccgta gaggcagcaa caccatcgct
     1681 aatatcgccg gcctttgtcc taagtgccac gagctcgtgc acaaggacgc cgatacagcc
     1741 gagtctctga cagagatgaa aaccggcctg atgaagaagt atggaggcac cagcgtgctg
     1801 aaccagatca tccccaagct ggtggaaacc ctggccgacc tgtttcctgg ccacttccac
     1861 gtgacaaacg gctggaacac caaggagttc agagagaaac atcatctgga aaaggatcac
     1921 gacgtggatg cttactgcat cgcctgcagc cacctgaagc ctgaggaaac cctggtggaa
     1981 actgagcctt tcgagatcct gcagttcaga aagcacaaca gagccatcat tcaccaccag
     2041 accgagcgga cgtacaagct ggacggcgtg acagtggcta aaaatagaaa gaaacggatg
     2101 gaacagaaaa ccgacagcct ggaagattgg tacgtggaca tggccaagga acacggcaaa
     2161 acacaggccg atgccatgcg gagcagactg accgtgatca agagcacacg gtattacaac
     2221 acccctggca gaatgatgcc aggcacagtt ttcctgtacg agggcaagag atacgtgatg
     2281 accggccaga ttacaaacgg caagtactac agagcctacg gccaggagaa gcggaacttc
     2341 cccgctgtca aggtgaggat tctgacaaag aacaccggac tagtctttgt ggcctagcta
     2401 gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact
     2461 atatccggat atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag
     2521 catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg
     2581 cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa
     2641 gctgtcaaac atgagaattc ttgaagacga aagggcctcg tgatacgcct atttttatag
     2701 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     2761 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2821 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     2881 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     2941 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3001 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3061 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtgt tgacgccggg
     3121 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3181 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3241 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3301 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3361 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgc agcaatggca
     3421 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3481 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3541 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     3601 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     3661 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     3721 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     3781 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     3841 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     3901 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     3961 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4021 agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag
     4081 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4141 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4201 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4261 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4321 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4381 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4441 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4501 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt tcctgcgtta
     4561 tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac cgctcgccgc
     4621 agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg
     4681 tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact ctcagtacaa
     4741 tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac gtgactgggt
     4801 catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4861 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4921 ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg
     4981 aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt tctccagaag
     5041 cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt cctgtttggt
     5101 cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac cgatgaaacg
     5161 agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac tggaacgttg
     5221 tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca ctcagggtca
     5281 atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc agcatcctgc
     5341 gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttaca gactttacga
     5401 aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca
     5461 gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa ggcaaccccg
     5521 ccagcctagc cgggtcctca acgacaggag cacgatcatg cgcacccgtg gccaggaccc
     5581 aacgctgccc gagatgcgcc gcgtgcggct gctggagatg gcggacgcga tggatatgtt
     5641 ctgccaaggg ttggtttgcg cattcacagt tctccgcaag aattgattgg ctccaattct
     5701 tggagtggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg
     5761 ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc gcctacaatc
     5821 catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac gatcagcggt
     5881 ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg tccctgatgg
     5941 tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat gccgccggaa
     6001 gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc cagcaagacg
     6061 tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc gaaacgtttg
     6121 gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa taccgcaagc
     6181 gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat gacccagagc
     6241 gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag tgcggcgacg
     6301 atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc
     6361 ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     6421 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     6481 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     6541 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     6601 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     6661 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     6721 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     6781 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     6841 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     6901 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     6961 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     7021 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     7081 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     7141 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     7201 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     7261 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     7321 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     7381 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     7441 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     7501 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg
     7561 ccattcgatg gtgt
//
