LOCUS       sequence-245459-        5285 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 5285)
  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 5285)
  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..5285
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..128
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(129..197,198..989)
                     /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(347..366)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1160..1748
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1649..1668
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2036..2057
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2072..2077,2078..2095,2096..2102)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2110..2126
                     /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     2115..2137
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2134..2150
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2134..2150
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2169..2189
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2171..2189
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        2627..2875
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2627..2647
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2798..2817
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     CDS             complement(join(3062..3775,3776..3778,3779..3781))
                     /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(3065..3775,3776..3778,3779..3781))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3107..3128)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3456..3475
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3715..3736
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3778..3787
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(3825..4088)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     4365..4384
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4641..4660)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4642..4660)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4670..4687)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4670..4686)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4679..4701)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4828..5283
                     /direction=RIGHT
                     /label=M13 ori
                     /note="M13 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4915..4934)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5125..5146
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
       61 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
      121 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
      181 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
      241 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
      301 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
      361 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
      421 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
      481 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
      541 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
      601 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
      661 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
      721 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
      781 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
      841 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      901 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      961 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     1021 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     1081 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     1141 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     1201 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     1261 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc
     1321 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     1381 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     1441 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     1501 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     1561 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     1621 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     1681 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     1741 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1801 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1861 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1921 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1981 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     2041 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
     2101 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     2161 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggtaccact agtatgcatc
     2221 ctgcaggacg cgtcctaccc actccctcta tgcgcgctcg ctcactcact cggccctgga
     2281 gaccaaaggt ctccagactg ccggcctctg gccggcaggg ccgagtgagt gagcgagcgc
     2341 gcatagaggg agtgggtacc tccatcatct aggtttgcca gatctgatat cggcgcgccc
     2401 gagtccaaca cccgtgggaa tcccatgggc accatggccc ctcgctccaa aaatgctttc
     2461 gcgtcgcgca gacactgctc ggtagtttcg gggatcagcg tttgagtaag agcccgcgtc
     2521 tgaaccctcc gcgccgcccc ggccccagtg gaaagacgcg caggcaaaac gcaccacgtg
     2581 acggagcgtg accgcgcgcc gagcgcgcgc caaggtcggg caggaagagg gcctatttcc
     2641 catgattcct tcatatttgc atatacgata caaggctgtt agagagataa ttagaattaa
     2701 tttgactgta aacacaaaga tattagtaca aaatacgtga cgtagaaagt aataatttct
     2761 tgggtagttt gcagttttaa aattatgttt taaaatggac tatcatatgc ttaccgtaac
     2821 ttgaaagtat ttcgatttct tggctttata tatcttgtgg aaaggacgaa acaccgccac
     2881 gttcaccttg ccccacgttt aagagctatg ctggaaacag catagcaagt ttaaataagg
     2941 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttg cggccgcctc
     3001 gagtgatcaa aaaaccaaca cacgcttcca atgaaaataa acgatccttt attgctagcc
     3061 tttacttgta cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca
     3121 ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag ggcggactgg gtgctcaggt
     3181 agtggttgtc gggcagcaac acggggccgt cgccgatggg ggtgttctgc tggtagtggt
     3241 cggcgagctg cacgctgccg tcctcgatgt tgtggcggat cttgaagttc accttgatgc
     3301 cgttcttctg cttgtcggcc atgatataga cgttgtggct gttgtagttg tactccagct
     3361 tgtgccccag gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca
     3421 ccagggtgtc gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga
     3481 agaagatggt gcgctcctgg acgtagcctt cgggcatggc ggacttgaag aagtcgtgct
     3541 gcttcatgtg gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga
     3601 gggtgggcca gggcacgggc agcttgccgg tggtgcagat gaacttcagg gtcagcttgc
     3661 cgtaggtggc atcgccctcg ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc
     3721 cgtccagctc gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca
     3781 tggtggcgac cggtggatcc cgggcccgcg gtacccagct tggaggtgca caccaatgtg
     3841 gtgaatggtc aaatggcgtt tattgtatcg agctaggcac ttaaatacaa tatctctgca
     3901 atgcggaatt cagtggttcg tccaatccat gtcagacccg tctgttgcct tcctaataag
     3961 gcacgatcgt accaccttac ttccaccaat cggcatgcac ggtgcttttt ctctccttgt
     4021 aaggcatgtt gctaactcat ccttaccatg ttgcaagact accaagagta tttgcataag
     4081 actacatttc cccctcccta tgcaaaagcg aaactactat atcctgaggg gactcctaac
     4141 cgcgtacaac cgaagccccg cttttcgcct aaacacaccc tagtcccctc agatacgcgt
     4201 atatctggcc cgtacatcgc gaagcagcgc aaaacgccta accctaagca gattcttcat
     4261 gcaattgtcg gtcaagcctt gccttgttgt agcttaaatt ttgctcgcgc actactcagc
     4321 gacctccaac acacaagcag ggagcagata ctggcttaac tatgcggcat cagagcagat
     4381 tgtactgaga gtgcaccata cggatctgcg atgataagct gtcaaacatg agaattggtc
     4441 gacgatatca gatctaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4501 gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg
     4561 cctcagtgag cgagcgagcg cgcagagagg gagtggcccc cccccccccc cccccctgca
     4621 ttctagagag ctccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg
     4681 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     4741 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     4801 agttgcgcag cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
     4861 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     4921 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     4981 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     5041 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5101 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     5161 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     5221 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     5281 tttag
//
