LOCUS       sequence-331222-        5259 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Used for guide cloning.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5259)
  AUTHORS   Fang H, Bygrave AM, Roth RH, Johnson RC, Huganir RL
  TITLE     An optimized CRISPR/Cas9 approach for precise genome editing in 
            neurons.
  JOURNAL   Elife. 2021 Mar 10;10. pii: 65202. doi: 10.7554/eLife.65202.
  PUBMED    33689678
REFERENCE   2  (bases 1 to 5259)
  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: "Elife."; 
            date: "2021-03-10"; volume: "10. pii: 65202. doi"; pages: " 
            10.7554/eLife.65202"
COMMENT     Sequence Label: pSG
FEATURES             Location/Qualifiers
     source          1..5259
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1682..1701
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1745..1993
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1745..1765
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1916..1935
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2012..2087
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2281..2300)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2346..2401
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2400..2419)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2762..2778)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2762..2778)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2775..2797)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2786..2802
                     /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(2810..2816,2817..2834,2835..2840))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2855..2876
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2935..3130
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2981..3116
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3136..3270
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3173..3192)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3227..3246
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3338..3355)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3509..4097)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3589..3608)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4268..5059,5060..5128))
                     /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     4891..4910
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5129..5233)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattcgc atgcggccgc
     1741 tagcgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     1801 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     1861 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     1921 tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa
     1981 aggacgaaac accgggtctt cgagaagacc tgttttagag ctagaaatag caagttaaaa
     2041 taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt tttgttttag
     2101 agctagaaat agcaagttaa aataaggcta gtccgttttt agcgcgtgcg ccaattctgc
     2161 agacaaatgg ctctagaggt ggtaccgata tcgagctcaa ttcactcctc aggtgcaggc
     2221 tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat accactgaga
     2281 tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc
     2341 tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc
     2401 actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta
     2461 gagtttggca acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg
     2521 tcatcagtat atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac
     2581 ttgaggttag atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa
     2641 attttcctta catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat
     2701 agctgtccct cttctcttat ggagatccct cgacctgcag cccaagcttg gcgtaatcat
     2761 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     2821 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     2881 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct
     2941 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     3001 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     3061 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     3121 cttttgcaaa aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3181 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3241 ctcatcaatg tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg
     3301 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3361 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3421 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3481 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3541 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3601 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3661 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
     3721 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3781 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3841 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     3901 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     3961 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4021 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4081 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4141 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4201 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4261 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4321 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4381 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4441 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4501 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4561 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4621 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4681 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4741 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4801 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4861 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     4921 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     4981 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5041 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5101 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5161 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5221 taggggttcc gcgcacattt ccccgaaaag tgccacctg
//
