LOCUS       sequence-249660-        5248 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expresses sgRNA HEK, SpyCas9 scaffold (F+E) and GFP (transfection
            marker).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5248)
  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 5248)
  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 HEK
FEATURES             Location/Qualifiers
     source          1..5248
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(join(435..1148,1149..1151,1152..1154))
                     /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(438..1148,1149..1151,1152..1154))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(480..501)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     829..848
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1088..1109
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1151..1160
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1198..1459)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     1736..1755
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     repeat_region   1828..1957
                     /label=AAV2 ITR
     primer_bind     complement(2001..2020)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2002..2020)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2030..2047)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2030..2046)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2039..2061)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2187..2642)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2324..2345)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2536..2555
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2669..2773
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2774..2842,2843..3634)
                     /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(2992..3011)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3805..4393
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4294..4313
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4681..4702
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4717..4722,4723..4740,4741..4747)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4755..4771
                     /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     4760..4782
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4779..4795
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4779..4795
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4814..4834
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4816..4834
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gcactgcggc tggaggtggg tttaagagct atgctggaaa cagcatagca
      301 agtttaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt
      361 ttgcggccgc ctcgagtgat caaaaaacca acacacgctt ccaatgaaaa taaacgatcc
      421 tttattgcta gcctttactt gtacagctcg tccatgccga gagtgatccc ggcggcggtc
      481 acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac
      541 tgggtgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc
      601 tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag
      661 ttcaccttga tgccgttctt ctgcttgtcg gccatgatat agacgttgtg gctgttgtag
      721 ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc
      781 tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg
      841 ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg
      901 aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcactgcac gccgtaggtc
      961 agggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc
     1021 agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg
     1081 ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg
     1141 cccttgctca ccatggtggc gaccggtgga tcccgggccc gcggtaccca gcttggaggt
     1201 gcacaccaat gtggtgaatg gtcaaatggc gtttattgta tcgagctagg cacttaaata
     1261 caatatctct gcaatgcgga attcagtggt tcgtccaatc catgtcagac ccgtctgttg
     1321 ccttcctaat aaggcacgat cgtaccacct tacttccacc aatcggcatg cacggtgctt
     1381 tttctctcct tgtaaggcat gttgctaact catcgttacc atgttgcaag actacaagag
     1441 tattgcataa gactacattt ccccctccct atgcaaaagc gaaactacta tatcctgagg
     1501 ggactcctaa ccgcgtacaa ccgaagcccc gcttttcgcc taaacacacc ctagtcccct
     1561 cagatacgcg tatatctggc ccgtacatcg cgaagcagcg caaaacgcct aaccctaagc
     1621 agattcttca tgcaattgtc ggtcaagcct tgccttgttg tagcttaaat tttgctcgcg
     1681 cactactcag cgacctccaa cacacaagca gggagcagat actggcttaa ctatgcggca
     1741 tcagagcaga ttgtactgag agtgcaccat acggatctgc gatgataagc tgtcaaacat
     1801 gagaattggt cgacgatatc agatctgagg aacccctagt gatggagttg gccactccct
     1861 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     1921 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc cccccctgca
     1981 ttctagagag ctccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     2041 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     2101 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     2161 agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt aatattttgt taaaattcgc
     2221 gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc
     2281 ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag
     2341 tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga
     2401 tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc
     2461 actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa
     2521 cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt
     2581 agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc
     2641 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2701 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2761 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2821 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2881 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2941 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3001 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     3061 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3121 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3181 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3241 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3301 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3361 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3421 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3481 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3541 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3601 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3661 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3721 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3781 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3841 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3901 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     3961 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4021 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4081 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4141 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4201 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4261 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4321 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4381 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4441 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4501 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4561 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     4621 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     4681 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     4741 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     4801 ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctgggta ccactagtat
     4861 gcatcctgca ggacgcgtcc tacccactcc ctctatgcgc gctcgctcac tcactcggcc
     4921 ctggagacca aaggtctcca gggccgagtg agtgagcgag cgcgcataga gggagtgggt
     4981 acctccatca tctggtttcc cagatctgat atcggcgcgc ccgagtccaa cacccgtggg
     5041 aatcccatgg gcaccatggc ccctcgctcc aaaaatgctt tcgcgtcgcg cagacactgc
     5101 tcggtagttt cggggatcag cgtttgagta agagcccgcg tctgaaccct ccgcgccgcc
     5161 ccggccccag tggaaagacg cgcaggcaaa acgcaccacg tgacggagcg tgaccgcgcg
     5221 ccgagcgcgc gccaaggtcg ggcaggaa
//
