LOCUS       sequence-479115-        7345 bp    DNA     circular SYN 21-JUL-2025
DEFINITION  OrufIscB-KRK mammalian expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7345)
  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 7345)
  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: pHS1742
FEATURES             Location/Qualifiers
     source          1..7345
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     612..631
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(850..954)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1029..1048)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1220..1599
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1600..1803
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1754..1774
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1848..1867
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1848..1866
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1902..1911
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1917..1937
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3741..3788
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             3795..3875
                     /codon_start=1
                     /product="three tandem HA epitope tags"
                     /label=3xHA
                     /translation="YPYDVPDYAYPYDVPDYAYPYDVPDYA"
     primer_bind     complement(3921..3938)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3927..4151
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4197..4625
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4284..4303)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4494..4515
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4634..4654)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4639..4968
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4819..4954
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4881..4900
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5035..5829
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5089..5108)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5699..5718
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6003..6124
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6040..6059)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6094..6113
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6173..6189)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6173..6189)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6186..6208)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6197..6213
                     /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)."
     promoter        complement(join(6221..6227,6228..6245,6246..6251))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6266..6287
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6404..6421)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6575..7163)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6655..6674)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7334..7345,1..780,781..849))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
ORIGIN
        1 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
       61 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
      121 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
      181 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
      241 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
      301 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
      361 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
      421 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
      481 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
      541 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
      601 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
      661 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
      721 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
      781 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
      841 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
      901 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
      961 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
     1021 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
     1081 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
     1141 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
     1201 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
     1261 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
     1321 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
     1381 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
     1441 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
     1501 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
     1561 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
     1621 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
     1681 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
     1741 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
     1801 gctctctggc taactagaga acccactgct tactggctta tcgaaattaa tacgactcac
     1861 tatagggaga cccaagctgg ctagcgttta aacttaagct tgccaccatg ggatctccaa
     1921 aaaagaaacg gaaggtggga tccagatacg tgtacgtgct ggatgtggac ggcaagcctc
     1981 tgatgcctac atgcaggttc ggcaaggtgc ggagaatgct gaaatctggc caagctaagg
     2041 ccgtggacac cctgcctttc accatccagc tgacctacaa gcccagaacc agaatcctgc
     2101 aacctgtgac cctgggccag gaccccggca gaaccaatat cggaatggcc gccgtgcggt
     2161 tcgacggcaa ggaactggga agattccact gcatcacaag aaacaaggaa atccctaagc
     2221 tgatggccga cagaatggca gccagaaagg cctcccggag aggcgagaga ctggccagaa
     2281 agcgcctggc ccggaaactg cacaccaccg ccaagcacct gaatggcaga atcctgccag
     2341 gatgtagcaa acccatcgcc gtgaaggaca tcatcaacac agagagcaga ttcaacaaca
     2401 gaatcctgac caaatgcaaa gtgtgcggca agaacacccc tctgagaaga aacgtgcggg
     2461 aactcctgct ggaaaacatc gtgcggtttc tgcctctgga aagcgagctg aaggaaacac
     2521 tcaagagaac catcctggaa ggccagcagg gcaatatcaa caagctgttc aggaaactta
     2581 aattcaatca aaaggattgg cctggcaaaa acctgaccga tatcgctaag aataagctgc
     2641 caggccggct gcccttctgc aaggagcact tcgccgagaa cgagaagttc accaccatcg
     2701 agaagagcac cttccggctg acccctaccg ccacccagct gctgcggacc cacatcaacc
     2761 tgtttagaaa gctgagcggc atcctgcccg tgaccgacgt ggccgtcgag ctgaacaagt
     2821 tcgcctttat gcagctggac aaccctgaga tgaagaagcg ggaaatcgac ttctgccacg
     2881 gcccactgtg cggcaccggc ggcctggaag ccgctgttaa ggagcagcag gatggaaaat
     2941 gcctgctgtg tggaaaagaa tctatcggcc actaccacca catcgtgcct aggtcccgta
     3001 gaggcagcaa caccatcgct aatatcgccg gcctttgtcc taagtgccac gagctcgtgc
     3061 acaaggacgc cgatacagcc gagtctctga cagagatgaa aaccggcctg atgaagaagt
     3121 atggaggcac cagcgtgctg aaccagatca tccccaagct ggtgcgcacc ctggccgacc
     3181 tgtttcctgg ccacttccac gtgacaaacg gctggaacac caaggagttc agagagaaac
     3241 atcatctgga aaaggatcac gacgtggatg cttactgcat cgcctgcagc cacctgaagc
     3301 ctgaggaaac cctggtggaa actgagcctt tcgagatcct gcagttcaga aagcacaaca
     3361 gagccatcat tcaccaccag accgagcgga cgtacaagct ggacggcgtg acagtggcta
     3421 aaaatagaaa gaaacggatg gaacagaaaa ccgacagcct ggaagattgg tacgtggaca
     3481 tggccaagga acacggcaaa acacaggccg atgccatgcg gagcagactg accgtgaaaa
     3541 agagcacacg gtattacaac acccctggca gaatgatgcc aggcacagtt ttcctgtacg
     3601 agggcaagag atacgtgatg accggccaga ttacaaacgg caagtactac agagcctacg
     3661 gccaggagaa gcggaacttc cccgctgtca aggtgaggat tctgacaaag aacaccggac
     3721 tagtctttgt ggccggatcc aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa
     3781 agaaaaaggg atcttaccca tacgatgttc cagattacgc ttacccatac gatgttccag
     3841 attacgctta cccatacgat gttccagatt acgcttgagg ccgctcgagt ctagagggcc
     3901 cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     3961 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     4021 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     4081 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     4141 gggctctatg gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc
     4201 gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac
     4261 acttgccagc gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt
     4321 cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc
     4381 tttacggcac ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc
     4441 gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact
     4501 cttgttccaa actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg
     4561 gattttgccg atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc
     4621 gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4681 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4741 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4801 ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc
     4861 catggctgac taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta
     4921 ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga
     4981 gcttgtatat ccattttcgg atctgatcaa gagacaggat gaggatcgtt tcgcatgatt
     5041 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat
     5101 gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag
     5161 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac
     5221 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac
     5281 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc
     5341 ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg
     5401 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag
     5461 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat
     5521 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag
     5581 gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc
     5641 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg
     5701 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg
     5761 ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag
     5821 ttcttctgag cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat
     5881 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     5941 gggacgccgg ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc
     6001 ccaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     6061 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     6121 cttatcatgt ctgtataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc
     6181 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     6241 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     6301 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     6361 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     6421 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     6481 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     6541 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     6601 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     6661 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     6721 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     6781 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     6841 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     6901 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     6961 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     7021 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     7081 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     7141 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     7201 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     7261 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     7321 cttggtctga cagttaccaa tgctt
//
