LOCUS       sequence-443125-        7461 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CMV-driven expression of enOgeuIscB and human U6-driven expression 
            of omega-RNA-v13 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-v13
FEATURES             Location/Qualifiers
     source          1..7461
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(125..229)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(304..323)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        495..874
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        875..1078
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1029..1049
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1123..1142
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1123..1141
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1177..1186
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1192..1212
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2710..2757
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     primer_bind     complement(2803..2820)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2809..3033
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3079..3507
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3166..3185)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3376..3397
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3516..3536)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3521..3850
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3701..3836
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3763..3782
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3917..4711
                     /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(3971..3990)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4581..4600
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4885..5006
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4922..4941)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4976..4995
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     5042..5064
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5056..5073
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     5057..5073
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(5302..5550)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(5360..5379)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5530..5550)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(5564..5580)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5564..5580)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5577..5599)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5588..5604
                     /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(5612..5618,5619..5636,5637..5642))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5657..5678
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5795..5812)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5966..6554)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6046..6065)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6725..7461,1..55,56..124))
                     /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     7348..7367
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
       61 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
      121 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
      181 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
      241 gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat ggtgcactct
      301 cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt
      361 ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag gcttgaccga
      421 caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc
      481 cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      541 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      601 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      661 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      721 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      781 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      841 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      901 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      961 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
     1021 cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctct
     1081 ctggctaact agagaaccca ctgcttactg gcttatcgaa attaatacga ctcactatag
     1141 ggagacccaa gctggctagc gtttaaactt aagcttgcca ccatgggatc tccaaaaaag
     1201 aaacggaagg tgggatctat ggccgtggtc tacgtgatct ctaagtccgg caaaccactg
     1261 atgcccacca ccagatgcgg ccacgtgcgg atcttgctca aggaaggcaa ggctagagtg
     1321 gtggaaagaa agcccttcac catccagctg acctacgagt ctgccgagga aacccagcct
     1381 ctggtgctgg gcattgaccc tggcagaacc aatatcggta tgagcgtggt gaccgagagc
     1441 ggagagagcg tgttcaacgc ccaaatcgag acaagaaaca aggacgtgcc taagctgatg
     1501 aaggacagaa agcagtatag gcgggcccac agaaggctga agcggcggtg caaaagacgg
     1561 cggagagcca aggctgccgg caccgccttc gaggagggcg aaaagcaaag actgctgcct
     1621 ggctgcaaga agcctattac atgcaagagc atccggaaca aggaagccag attcaacaac
     1681 cgcaagcggc ctaagggatg gctgacacct accgccaatc acctgctggt cacccacctg
     1741 aacgtggtta agaaggtgca gaagatcctg cccgtggcca aggtggtgct ggagctgaat
     1801 cggttcagct ttatggccat gaacaacccc aaggttcaga gatggcagta ccagcggggc
     1861 cctctgtacg gcaaaggcag cgtggaagag gccgtgtcca tgcagcagga tggacactgc
     1921 ctgttttgta aacacggcat cgaccactac caccacgtgg tccccagaag aaagaacggc
     1981 agcgagacac tggaaaacag agtgggcctt tgtgaagaac accatagact ggtgcacaca
     2041 gataaggagt gggaggccag actggccagc aagaagtctg gaatgaacaa gaaataccac
     2101 gccctgtctg tgctgaacca gatcatcccc tacctggctg atcagctggc cgatatgttc
     2161 cccggaaatt tctgcgtgac aagcggccgg gacaccaagc tgtttcggga agagcatggc
     2221 atccctaagg accactacct ggacgcctac tgcatcgcct gcagcgcact gaccgacgcc
     2281 aagaaggtga gcagccctaa gggcagacca tacatggtga gacagttcag aaggcatgat
     2341 agacaggcct gtcacaaggc caatctgaac cggagctact acatgggcgg caagaaggtt
     2401 gccaccaacc ggcacaaagc tatggaccag aaaactgaca gcctggaaga gtacagagcc
     2461 gctcacagcg ccgctgacgt gtctaaactg accgtgaagc acccttctgc tcagtacaag
     2521 gatatgagca gaatcatgcc tggcagcatc ctggtgagcg gcgagggcaa actgttcaca
     2581 ctgagcagat ctgagggaag aaacaaaggc caggtgaact acttcgtgtc caccgagggc
     2641 atcaagtact gggccagaaa gtgccagtat ctgcggaaca acggcggact gcagatctac
     2701 gtgggatcca aaaggccggc ggccacgaaa aaggccggcc aggcaaaaaa gaaaaagtga
     2761 ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta
     2821 gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca
     2881 ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc
     2941 attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata
     3001 gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg
     3061 gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     3121 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct
     3181 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     3241 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg
     3301 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     3361 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
     3421 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc
     3481 tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg
     3541 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3601 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     3661 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3721 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3781 ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3841 cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg
     3901 atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg
     3961 ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc
     4021 cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg
     4081 tgccctgaat gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt
     4141 tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg
     4201 cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat
     4261 catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca
     4321 ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca
     4381 ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa
     4441 ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa
     4501 tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc
     4561 ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga
     4621 atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc
     4681 cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac
     4741 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     4801 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     4861 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     4921 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     4981 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     5041 tcccagtcac gacgttgtaa aacgacggcc agtgaattcg agctcggtac caaaaaaaac
     5101 tctctctgtt tccagagagg tgattgttcc ccctggctta tctgattttc atcaactgac
     5161 atccgctatg cagttccggg gactggggaa atcccagggt gcgtccggcc tttcggcctt
     5221 acgaatgtct cagcctgtgc tctttcaacc ttttcaagtt ggaagagcct gagacgatct
     5281 cgttcactct tttcgtctct cggtgtttcg tcctttccac aagatatata aagccaagaa
     5341 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
     5401 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
     5461 tgtgtttaca gtcaaattaa ttccaattat ctctctaaca gccttgtatc gtatatgcaa
     5521 atatgaagga atcatgggaa ataggccctc tggcgtaatc atggtcatag ctgtttcctg
     5581 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
     5641 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     5701 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     5761 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     5821 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5881 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5941 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     6001 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     6061 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     6121 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     6181 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     6241 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     6301 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     6361 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     6421 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     6481 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     6541 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     6601 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     6661 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     6721 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6781 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6841 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6901 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6961 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     7021 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     7081 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     7141 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     7201 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     7261 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     7321 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     7381 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     7441 gatccagttc gatgtaaccc a
//
