LOCUS       sequence-249650-        5242 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Co-expresses sgRNA HBB, SpyCas9 scaffold (F+E) and GFP (transfection
            marker).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5242)
  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 5242)
  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 HBB
FEATURES             Location/Qualifiers
     source          1..5242
                     /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(485..1198,1199..1201,1202..1204))
                     /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(488..1198,1199..1201,1202..1204))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(530..551)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     879..898
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1138..1159
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1201..1210
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1248..1509)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     1786..1805
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     repeat_region   1878..2007
                     /label=AAV2 ITR
     primer_bind     complement(2044..2063)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2045..2063)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2073..2090)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2073..2089)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2082..2104)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2230..2685)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2367..2388)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2579..2598
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2712..2816
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2817..2885,2886..3677)
                     /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(3035..3054)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3848..4436
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4337..4356
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    4724..4745
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4760..4765,4766..4783,4784..4790)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4798..4814
                     /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     4803..4825
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4822..4838
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4822..4838
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4857..4877
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4859..4877
                     /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 gaaacaccgc
      301 cacgttcacc ttgccccacg tttaagagct atgctggaaa cagcatagca agtttaaata
      361 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgcggccgc
      421 ctcgagtgat caaaaaacca acacacgctt ccaatgaaaa taaacgatcc tttattgcta
      481 gcctttactt gtacagctcg tccatgccga gagtgatccc ggcggcggtc acgaactcca
      541 gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagggcggac tgggtgctca
      601 ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt
      661 ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg gatcttgaag ttcaccttga
      721 tgccgttctt ctgcttgtcg gccatgatat agacgttgtg gctgttgtag ttgtactcca
      781 gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt
      841 tcaccagggt gtcgccctcg aacttcacct cggcgcgggt cttgtagttg ccgtcgtcct
      901 tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat ggcggacttg aagaagtcgt
      961 gctgcttcat gtggtcgggg tagcggctga agcactgcac gccgtaggtc agggtggtca
     1021 cgagggtggg ccagggcacg ggcagcttgc cggtggtgca gatgaacttc agggtcagct
     1081 tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct gaacttgtgg ccgtttacgt
     1141 cgccgtccag ctcgaccagg atgggcacca ccccggtgaa cagctcctcg cccttgctca
     1201 ccatggtggc gaccggtgga tcccgggccc gcggtaccca gcttggaggt gcacaccaat
     1261 gtggtgaatg gtcaaatggc gtttattgta tcgagctagg cacttaaata caatatctct
     1321 gcaatgcgga attcagtggt tcgtccaatc catgtcagac ccgtctgttg ccttcctaat
     1381 aaggcacgat cgtaccacct tacttccacc aatcggcatg cacggtgctt tttctctcct
     1441 tgtaaggcat gttgctaact catcgttacc atgttgcaag actacaagag tattgcataa
     1501 gactacattt ccccctccct atgcaaaagc gaaactacta tatcctgagg ggactcctaa
     1561 ccgcgtacaa ccgaagcccc gcttttcgcc taaacacacc ctagtcccct cagatacgcg
     1621 tatatctggc ccgtacatcg cgaagcagcg caaaacgcct aaccctaagc agattcttca
     1681 tgcaattgtc ggtcaagcct tgccttgttg tagcttaaat tttgctcgcg cactactcag
     1741 cgacctccaa cacacaagca gggagcagat actggcttaa ctatgcggca tcagagcaga
     1801 ttgtactgag agtgcaccat acggatctgc gatgataagc tgtcaaacat gagaattggt
     1861 cgacgatatc agatctgagg aacccctagt gatggagttg gccactccct ctctgcgcgc
     1921 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
     1981 ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc gcattctaga gagctccaat
     2041 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     2101 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     2161 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     2221 ggcgaatgga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     2281 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     2341 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     2401 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     2461 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     2521 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     2581 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     2641 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc
     2701 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     2761 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     2821 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     2881 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     2941 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3001 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     3061 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3121 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3181 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     3241 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     3301 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     3361 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     3421 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     3481 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     3541 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     3601 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     3661 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     3721 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     3781 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     3841 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     3901 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     3961 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     4021 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     4081 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     4141 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     4201 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     4261 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     4321 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     4381 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     4441 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     4501 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     4561 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc
     4621 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     4681 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     4741 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     4801 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagcgcgcaa
     4861 ttaaccctca ctaaagggaa caaaagctgg gtaccactag tatgcatcct gcaggacgcg
     4921 tcctacccac tccctctatg cgcgctcgct cactcactcg gccctggaga cctttggtct
     4981 ccagggccga gtgagtgagc gagcgcgcat agagggagtg ggtacctcca tcatctggtt
     5041 tcccagatct gatatcggcg cgcccgagtc caacacccgt gggaatccca tgggcaccat
     5101 ggcccctcgc tccaaaaatg ctttcgcgtc gcgcagacac tgctcggtag tttcggggat
     5161 cagcgtttga gtaagagccc gcgtctgaac cctccgcgcc gccccggccc cagtggaaag
     5221 acgcgcaggc aaaacgcacc ac
//
