LOCUS       sequence-249653-        5093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expresses sgRNA EMX1, SpyCas9 scaffold (F+E) and GFP 
            (transfection marker).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5093)
  AUTHORS   Aschenbrenner S, Kallenberger SM, Hoffmann MD, Huck A, Eils R, 
            Niopek D
  TITLE     Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 
            target specificity.
  JOURNAL   Sci Adv. 2020 Feb 5;6(6):eaay0187. doi: 10.1126/sciadv.aay0187. 
            eCollection 2020 Feb.
  PUBMED    32076642
REFERENCE   2  (bases 1 to 5093)
  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: "Sci Adv."; 
            date: "2020-02-5"; volume: "6(6):eaay0187. doi"; pages: " 
            10.1126/sciadv.aay0187. eCollection 2020 Feb"
COMMENT     Sequence Label: sgRNA EMX1
FEATURES             Location/Qualifiers
     source          1..5093
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        50..298
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     50..70
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     221..240
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(join(484..1197,1198..1200,1201..1203))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(487..1197,1198..1200,1201..1203))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(529..550)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     878..897
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1137..1158
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1200..1209
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1247..1508)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     1785..1804
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1924..1941)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1924..1940)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1933..1955)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2081..2536)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2218..2239)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2430..2449
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2563..2667
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2668..2736,2737..3528)
                     /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     complement(2886..2905)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3699..4287
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4188..4207
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4575..4596
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4611..4616,4617..4634,4635..4641)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4649..4665
                     /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)."
     primer_bind     4654..4676
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4673..4689
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4673..4689
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4708..4728
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4710..4728
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gtgacggagc gtgaccgcgc gccgagcgcg cgccaaggtc gggcaggaag agggcctatt
       61 tcccatgatt ccttcatatt tgcatatacg atacaaggct gttagagaga taattagaat
      121 taatttgact gtaaacacaa agatattagt acaaaatacg tgacgtagaa agtaataatt
      181 tcttgggtag tttgcagttt taaaattatg ttttaaaatg gactatcata tgcttaccgt
      241 aacttgaaag tatttcgatt tcttggcttt atatatcttg tggaaaggac gaaacaccga
      301 gtccgagcag aagaagaagt ttaagagcta tgctggaaac agcatagcaa gtttaaataa
      361 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgcggccgcc
      421 tcgagtgatc aaaaaaccaa cacacgcttc caatgaaaat aaacgatcct ttattgctag
      481 cctttacttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca cgaactccag
      541 caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact gggtgctcag
      601 gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct gctggtagtg
      661 gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt tcaccttgat
      721 gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt tgtactccag
      781 cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt
      841 caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt
      901 gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg
      961 ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca gggtggtcac
     1021 gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca gggtcagctt
     1081 gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc cgtttacgtc
     1141 gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc ccttgctcac
     1201 catggtggcg accggtggat cccgggcccg cggtacccag cttggaggtg cacaccaatg
     1261 tggtgaatgg tcaaatggcg tttattgtat cgagctaggc acttaaatac aatatctctg
     1321 caatgcggaa ttcagtggtt cgtccaatcc atgtcagacc cgtctgttgc cttcctaata
     1381 aggcacgatc gtaccacctt acttccacca atcggcatgc acggtgcttt ttctctcctt
     1441 gtaaggcatg ttgctaactc atcgttacca tgttgcaaga ctacaagagt attgcataag
     1501 actacatttc cccctcccta tgcaaaagcg aaactactat atcctgaggg gactcctaac
     1561 cgcgtacaac cgaagccccg cttttcgcct aaacacaccc tagtcccctc agatacgcgt
     1621 atatctggcc cgtacatcgc gaagcagcgc aaaacgccta accctaagca gattcttcat
     1681 gcaattgtcg gtcaagcctt gccttgttgt agcttaaatt ttgctcgcgc actactcagc
     1741 gacctccaac acacaagcag ggagcagata ctggcttaac tatgcggcat cagagcagat
     1801 tgtactgaga gtgcaccata cggatctgcg atgataagct gtcaaacatg agaattggtc
     1861 gacgatatca gatctgagga acccctagtg nantggagtt ggccactccc tctctgcgcg
     1921 ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     1981 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     2041 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg aaattgtaag cgttaatatt
     2101 ttgttaaaat tcgcgttaaa tttttgttaa atcagctcat tttttaacca ataggccgaa
     2161 atcggcaaaa tcccttataa atcaaaagaa tagaccgaga tagggttgag tgttgttcca
     2221 gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg gcgaaaaacc
     2281 gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt tttggggtcg
     2341 aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag agcttgacgg
     2401 ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg
     2461 gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg
     2521 ccgctacagg gcgcgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     2581 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     2641 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     2701 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     2761 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     2821 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     2881 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     2941 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     3001 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     3061 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     3121 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     3181 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     3241 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     3301 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     3361 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     3421 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     3481 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     3541 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     3601 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     3661 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     3721 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     3781 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     3841 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     3901 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     3961 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     4021 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     4081 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     4141 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     4201 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     4261 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     4321 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     4381 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     4441 agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg
     4501 cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt
     4561 gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt
     4621 atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac
     4681 agctatgacc atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg
     4741 ggtaccacta gtatgcatcc tgcaggacgc gtcctaccca ctccctctat gcgcgctcgc
     4801 tcactcactc ggccctggag accaaaggtc tccagggccg agtgagtgag cgagcgcgca
     4861 tagagggagt gggtacctcc atcatctggt ttcccagatc tgatatcggc gcgcccgagt
     4921 ccaacacccg tgggaatccc atgggcacca tggcccctcg ctccaaaaat gctttcgcgt
     4981 cgcgcagaca ctgctcggta gtttcgggga tcagcgtttg agtaagagcc cgcgtctgaa
     5041 ccctccgcgc cgccccggcc ccagtggaaa gacgcgcagg caaaacgcac cac
//
