LOCUS       sequence-431656-        6421 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid encodes sgRNA that target EGFP with stretch sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6421)
  AUTHORS   Ui-Tei K, Maruyama S, Nakano Y
  TITLE     Enhancement of single guide RNA transcription for efficient 
            CRISPR/Cas-based genomic engineering.
  JOURNAL   Genome. 2017 Jun;60(6):537-545. doi: 10.1139/gen-2016-0127. Epub 
            2017 Jan 26.
  PUBMED    28177825
REFERENCE   2  (bases 1 to 6421)
  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"; doi: 
            "10.1139/gen-2016-0127"; journalName: "Genome"; date: "2017-06"; 
            volume: "60"; issue: "6"; pages: "537-545"
COMMENT     Sequence Label: pSuperCRISPR-sgRNA-EGFP12-TTTA
FEATURES             Location/Qualifiers
     source          1..6421
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     1421..1437
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1422..1438
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        complement(1453..1528)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(1556..1770)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(1616..1635)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     promoter        1781..2280
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1816..1839)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2213..2232
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2305..2904
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2305..2324)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2801..2821
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     protein_bind    3490..3506
                     /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)."
     promoter        complement(join(3514..3520,3521..3538,3539..3544))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3559..3580
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3697..3714)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3868..4456)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3948..3967)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4627..5418,5419..5487))
                     /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     5250..5269
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5488..5592)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5660..5678
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5716..5738)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5838..5857
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6051..6073
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6201..6220
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6420..6421,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg accccccagc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatcgatctc tcgaggtcga cggtatcgat
     1441 aagcttaaaa aagcaccgac tcggtgccac tttttcaagt tgataacgga ctagccttat
     1501 ttaaacttgc tatttctagc tcttaaaccg gtgaacagct cctcgcccgg gatctgtggt
     1561 ctcatacaga acttataaga ttcccaaatc caaagacatt tcacgtttat ggtgatttcc
     1621 cagaacacat agcgacatgc aaatattgca gggcgccact cccctgtccc tcacagccat
     1681 cttcctgcca gggcgcacgc gcgctgggtg ttcccgccta gtgacactgg gcccgcgatt
     1741 ccttggagcg ggttgatgac gtcagcgttc gaattctacc gggtagggga ggcgcttttc
     1801 ccaaggcagt ctggagcatg cgctttagca gccccgctgg gcacttggcg ctacacaagt
     1861 ggcctctggc ctcgcacaca ttccacatcc accggtaggc gccaaccggc tccgttcttt
     1921 ggtggcccct tcgcgccacc ttctactcct cccctagtca ggaagttccc ccccgccccg
     1981 cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac gtctcactag tctcgtgcag
     2041 atggacagca ccgctgagca atggaagcgg gtaggccttt ggggcagcgg ccaatagcag
     2101 ctttgctcct tcgctttctg ggctcagagg ctgggaaggg gtgggtccgg gggcgggctc
     2161 aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct ccggaggccc ggcattctgc
     2221 acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt cctcatctcc gggcctttcg
     2281 acctgcagcc caagctagct taccatgacc gagtacaagc ccacggtgcg cctcgccacc
     2341 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     2401 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     2461 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     2521 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     2581 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     2641 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     2701 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     2761 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     2821 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     2881 atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg
     2941 agcgcacgac cccatgcatc gataaaataa aagattttat ttagtctcca gaaaaagggg
     3001 ggaatgaaag accccacctg taggtttggc aagctagaga accatcagat gtttccaggg
     3061 tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca gttcgcttct
     3121 cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca acccctcact
     3181 cggcgcgcca gtcctccgat agactgcgtc gcccgggtac ccgtgtatcc aataaaccct
     3241 cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc tctgagtgat
     3301 tgactacccg tcagcggggg tctttcatgg gtaacagttt cttgaagttg gagaacaaca
     3361 ttctgagggt aggagtcgaa tattaagtaa tcctgactca attagccact gttttgaatc
     3421 cacatactcc aatactcctg aaatagttca ttatggacag cgcagaaaga gctggggaga
     3481 attgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3541 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3601 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     3661 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3721 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3781 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3841 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3901 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3961 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4021 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4081 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4141 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4201 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4261 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4321 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4381 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4441 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4501 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     4561 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     4621 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     4681 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     4741 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     4801 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     4861 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     4921 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     4981 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5041 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5101 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5161 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5221 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5281 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5341 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5401 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5461 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     5521 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     5581 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat
     5641 catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg
     5701 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     5761 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     5821 gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg
     5881 tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag
     5941 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     6001 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     6061 acgttgtaaa acgacggcgc aaggaatggt gcatgcaagg agatggcgcc caacagtccc
     6121 ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag cccgaagtgg
     6181 cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac cgcacctgtg
     6241 gcgccggtga tgccggccac gatgcgtccg gcgtagaggc gattagtcca atttgttaaa
     6301 gacaggatat cagtggtcca ggctctagtt ttgactcaac aatatcacca gctgaagcct
     6361 atagagtacg agccatagat aaaataaaag attttattta gtctccagaa aaagggggga
     6421 a
//
