LOCUS       sequence-342406-        5265 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 5265)
  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 5265)
  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..5265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     intron          160..1176
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1100..1122
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1184..1203
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1249..1497
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1249..1269
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1420..1439
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1516..1591
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1785..1804)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1850..1905
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(1904..1923)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2266..2282)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2266..2282)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2279..2301)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2290..2306
                     /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(2314..2320,2321..2338,2339..2344))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2359..2380
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2439..2634
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2485..2620
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2640..2774
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2677..2696)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2731..2750
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2842..2859)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3013..3601)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3093..3112)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3772..4563,4564..4632))
                     /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     4395..4414
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4633..4737)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        4768..5147
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        join(5149..5265,1..159)
                     /label=chicken beta-actin promoter
ORIGIN
        1 gggcgcgcgc caggcggggc ggggcggggc gaggggcggg gcggggcgag gcggagaggt
       61 gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc cttttatggc gaggcggcgg
      121 cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg gagtcgctgc gcgctgcctt
      181 cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc cccggctctg actgaccgcg
      241 ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa ttagcgcttg
      301 gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa gccttgaggg gctccgggag
      361 ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg tgtgtgtgtg cgtggggagc
      421 gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct gcgggcgcgg cgcggggctt
      481 tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg gcggtgcccc gcggtgcggg
      541 gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg
      601 ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc cccctccccg agttgctgag
      661 cacggcccgg cttcgggtgc ggggctccgt acggggcgtg gcgcggggct cgccgtgccg
      721 ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg ggccgcctcg ggccggggag
      781 ggctcggggg aggggcgcgg cggcccccgg agcgccggcg gctgtcgagg cgcggcgagc
      841 cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa
      901 atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc cctctagcgg gcgcggggcg
      961 aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg gccttcgtgc gtcgccgcgc
     1021 cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc ggggggacgg ctgccttcgg
     1081 gggggacggg gcagggcggg gttcggcttc tggcgtgtga ccggcggctc tagagcctct
     1141 gctaaccatg ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt
     1201 gtgctgtctc atcattttgg caaagaattc gcatgcggcc gcgctagcga gggcctattt
     1261 cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt
     1321 aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt
     1381 cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta
     1441 acttgaaagt atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg
     1501 tcttcgagaa gacctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta
     1561 tcaacttgaa aaagtggcac cgagtcggtg cttttttgtt ttagagctag aaatagcaag
     1621 ttaaaataag gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa atggctctag
     1681 aggtggtacc gatatcgagc tcaattcact cctcaggtgc aggctgccta tcagaaggtg
     1741 gtggctggtg tggccaatgc cctggctcac aaataccact gagatctttt tccctctgcc
     1801 aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta ataaaggaaa
     1861 tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg aaggacatat
     1921 gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt ggcaacatat
     1981 gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca gtatatgaaa
     2041 cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt
     2101 tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt
     2161 ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc
     2221 ttatggagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc
     2281 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     2341 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     2401 cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc
     2461 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     2521 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     2581 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta
     2641 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     2701 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     2761 atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     2821 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     2881 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     2941 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3001 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3061 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3121 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3181 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3241 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3301 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3361 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3421 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     3481 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     3541 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     3601 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     3661 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3721 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     3781 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3841 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3901 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     3961 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4021 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4081 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4141 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     4201 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     4261 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     4321 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     4381 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4441 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4501 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4561 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     4621 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4681 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     4741 atttccccga aaagtgccac ctgggtcgac attgattatt gactagttat taatagtaat
     4801 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
     4861 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
     4921 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
     4981 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
     5041 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
     5101 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc gaggtgagcc
     5161 ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt ttgtatttat
     5221 ttatttttta attattttgt gcagcgatgg gggcgggggg ggggg
//
