LOCUS       sequence-249646-        5091 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expresses sgRNA AAVS1, SpyCas9 scaffold (F+E) and GFP 
            (transfection marker).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5091)
  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 5091)
  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 AAVS1
FEATURES             Location/Qualifiers
     source          1..5091
                     /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(1922..1939)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1922..1938)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1931..1953)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2079..2534)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2216..2237)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2428..2447
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2561..2665
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2666..2734,2735..3526)
                     /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(2884..2903)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3697..4285
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4186..4205
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4573..4594
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4609..4614,4615..4632,4633..4639)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4647..4663
                     /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     4652..4674
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4671..4687
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4671..4687
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4706..4726
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4708..4726
                     /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 gaaacaccgg
      301 gagggagagc ttggcagggt 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 atggagttgg ccactccctc tctgcgcgct
     1921 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     1981 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     2041 gcccttccca acagttgcgc agcctgaatg gcgaatggaa attgtaagcg ttaatatttt
     2101 gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat
     2161 cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt
     2221 ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt
     2281 ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag
     2341 gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg
     2401 aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc
     2461 gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc
     2521 gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     2581 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     2641 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     2701 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     2761 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     2821 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     2881 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     2941 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     3001 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     3061 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     3121 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     3181 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     3241 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     3301 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     3361 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     3421 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     3481 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     3541 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     3601 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     3661 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     3721 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     3781 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     3841 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     3901 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     3961 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     4021 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     4081 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     4141 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     4201 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     4261 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     4321 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     4381 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     4441 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     4501 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     4561 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     4621 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     4681 ctatgaccat gattacgcca agcgcgcaat taaccctcac taaagggaac aaaagctggg
     4741 taccactagt atgcatcctg caggacgcgt cctacccact ccctctatgc gcgctcgctc
     4801 actcactcgg ccctggagac ctttggtctc cagggccgag tgagtgagcg agcgcgcata
     4861 gagggagtgg gtacctccat catctggttt cccagatctg atatcggcgc gcccgagtcc
     4921 aacacccgtg ggaatcccat gggcaccatg gcccctcgct ccaaaaatgc tttcgcgtcg
     4981 cgcagacact gctcggtagt ttcggggatc agcgtttgag taagagcccg cgtctgaacc
     5041 ctccgcgccg ccccggcccc agtggaaaga cgcgcaggca aaacgcacca c
//
