LOCUS       sequence-443121-        7461 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-driven expression of enOgeuIscB and human U6-driven expression 
            of omega-RNA-v2 scaffold with BsmBI for guide cloing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7461)
  AUTHORS   Yan H, Tan X, Zou S, Sun Y, Ke A, Tang W
  TITLE     Assessing and engineering the IscB-omegaRNA system for programmed 
            genome editing.
  JOURNAL   Nat Chem Biol. 2024 Jul 8. doi: 10.1038/s41589-024-01669-3.
  PUBMED    38977787
REFERENCE   2  (bases 1 to 7461)
  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 Chem 
            Biol. 2024 Jul 8. doi: 10.1038/s41589-024-01669-3."
COMMENT     Sequence Label: enOgeuIscB with omega-RNA-v2
FEATURES             Location/Qualifiers
     source          1..7461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(117..221)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(296..315)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        487..866
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        867..1070
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1021..1041
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1115..1134
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1115..1133
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1169..1178
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1184..1204
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2702..2749
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(2795..2812)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2801..3025
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3071..3499
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3158..3177)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3368..3389
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3508..3528)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3513..3842
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3693..3828
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3755..3774
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3909..4703
                     /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(3963..3982)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4573..4592
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4877..4998
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4914..4933)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4968..4987
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5034..5056
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5048..5065
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5049..5065
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(5294..5542)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5352..5371)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5522..5542)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5556..5572)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5556..5572)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5569..5591)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5580..5596
                     /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(5604..5610,5611..5628,5629..5634))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5649..5670
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5787..5804)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5958..6546)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6038..6057)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6717..7461,1..47,48..116))
                     /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"
     primer_bind     7340..7359
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
       61 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
      121 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
      181 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
      241 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
      301 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      361 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      421 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      481 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      541 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      601 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      661 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      721 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      781 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      841 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      901 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      961 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
     1021 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa
     1081 ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc
     1141 aagctggcta gcgtttaaac ttaagcttgc caccatggga tctccaaaaa agaaacggaa
     1201 ggtgggatct atggccgtgg tctacgtgat ctctaagtcc ggcaaaccac tgatgcccac
     1261 caccagatgc ggccacgtgc ggatcttgct caaggaaggc aaggctagag tggtggaaag
     1321 aaagcccttc accatccagc tgacctacga gtctgccgag gaaacccagc ctctggtgct
     1381 gggcattgac cctggcagaa ccaatatcgg tatgagcgtg gtgaccgaga gcggagagag
     1441 cgtgttcaac gcccaaatcg agacaagaaa caaggacgtg cctaagctga tgaaggacag
     1501 aaagcagtat aggcgggccc acagaaggct gaagcggcgg tgcaaaagac ggcggagagc
     1561 caaggctgcc ggcaccgcct tcgaggaggg cgaaaagcaa agactgctgc ctggctgcaa
     1621 gaagcctatt acatgcaaga gcatccggaa caaggaagcc agattcaaca accgcaagcg
     1681 gcctaaggga tggctgacac ctaccgccaa tcacctgctg gtcacccacc tgaacgtggt
     1741 taagaaggtg cagaagatcc tgcccgtggc caaggtggtg ctggagctga atcggttcag
     1801 ctttatggcc atgaacaacc ccaaggttca gagatggcag taccagcggg gccctctgta
     1861 cggcaaaggc agcgtggaag aggccgtgtc catgcagcag gatggacact gcctgttttg
     1921 taaacacggc atcgaccact accaccacgt ggtccccaga agaaagaacg gcagcgagac
     1981 actggaaaac agagtgggcc tttgtgaaga acaccataga ctggtgcaca cagataagga
     2041 gtgggaggcc agactggcca gcaagaagtc tggaatgaac aagaaatacc acgccctgtc
     2101 tgtgctgaac cagatcatcc cctacctggc tgatcagctg gccgatatgt tccccggaaa
     2161 tttctgcgtg acaagcggcc gggacaccaa gctgtttcgg gaagagcatg gcatccctaa
     2221 ggaccactac ctggacgcct actgcatcgc ctgcagcgca ctgaccgacg ccaagaaggt
     2281 gagcagccct aagggcagac catacatggt gagacagttc agaaggcatg atagacaggc
     2341 ctgtcacaag gccaatctga accggagcta ctacatgggc ggcaagaagg ttgccaccaa
     2401 ccggcacaaa gctatggacc agaaaactga cagcctggaa gagtacagag ccgctcacag
     2461 cgccgctgac gtgtctaaac tgaccgtgaa gcacccttct gctcagtaca aggatatgag
     2521 cagaatcatg cctggcagca tcctggtgag cggcgagggc aaactgttca cactgagcag
     2581 atctgaggga agaaacaaag gccaggtgaa ctacttcgtg tccaccgagg gcatcaagta
     2641 ctgggccaga aagtgccagt atctgcggaa caacggcgga ctgcagatct acgtgggatc
     2701 caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt gaggccgctc
     2761 gagtctagag ggcccgttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     2821 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     2881 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     2941 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     3001 gctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     3061 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3121 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     3181 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     3241 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     3301 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     3361 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     3421 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     3481 caaaaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     3541 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     3601 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     3661 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3721 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3781 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3841 aaaagctccc gggagcttgt atatccattt tcggatctga tcaagagaca ggatgaggat
     3901 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     3961 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     4021 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     4081 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     4141 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     4201 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
     4261 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     4321 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     4381 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     4441 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     4501 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     4561 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     4621 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     4681 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac
     4741 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4801 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4861 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4921 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4981 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gctcccagtc
     5041 acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt accaaaaaaa actctctctg
     5101 tttccagaga ggtgattgtt ccccctggct tatctgattt tcatcaactg acatccgcta
     5161 tgcagttccg gggactgggg aaatcccagg gtgcgtccgg cctttcggcc ttacgaatgt
     5221 ctcagcctgt gctctttcaa ccttttcaag ttgaaagagc ctgagacgat ctcgttcact
     5281 cttttcgtct ctcggtgttt cgtcctttcc acaagatata taaagccaag aaatcgaaat
     5341 actttcaagt tacggtaagc atatgatagt ccattttaaa acataatttt aaaactgcaa
     5401 actacccaag aaattattac tttctacgtc acgtattttg tactaatatc tttgtgttta
     5461 cagtcaaatt aattccaatt atctctctaa cagccttgta tcgtatatgc aaatatgaag
     5521 gaatcatggg aaataggccc tctggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     5581 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     5641 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5701 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5761 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5821 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5881 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5941 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     6001 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     6061 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     6121 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6181 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6241 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6301 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6361 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     6421 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6481 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6541 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6601 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6661 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6721 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6781 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6841 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6901 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6961 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     7021 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     7081 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     7141 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7201 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7261 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7321 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7381 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7441 tcgatgtaac ccactcgtgc a
//
