LOCUS       sequence-397514-        5479 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Positive control plasmid expressing mouse Tubb3 GS-gRNA (with 
            hSyn-Cre).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5479)
  AUTHORS   Gao Y, Hisey E, Bradshaw TWA, Erata E, Brown WE, Courtland JL, Uezu 
            A, Xiang Y, Diao Y, Soderling SH
  TITLE     Plug-and-Play Protein Modification Using Homology-Independent 
            Universal Genome Engineering.
  JOURNAL   Neuron. 2019 Aug 21;103(4):583-597.e8. doi: 
            10.1016/j.neuron.2019.05.047. Epub 2019 Jul 1.
  PUBMED    31272828
REFERENCE   2  (bases 1 to 5479)
  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: 
            "10.1016/j.neuron.2019.05.047"; journalName: "Neuron"; date: 
            "2019-08-21- 21"; volume: "103"; issue: "4"; pages: "583-597.e8"
COMMENT     Sequence Label: pHiUGE GS-gRNA (hSyn-Cre) mTubb3
FEATURES             Location/Qualifiers
     source          1..5479
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        145..393
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     145..165
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     316..335
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        414..489
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        523..970
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             993..1013
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2067..2655
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2120..2140)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2538..2549)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   2742..2882
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2742..2871
                     /label=AAV2 ITR
     rep_origin      2957..3412
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3044..3063)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3254..3275
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3429..3448)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3548..3570
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3608..3626)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3694..3798
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3799..3867,3868..4659)
                     /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(4017..4036)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4830..5418
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5319..5338
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtgagggc ctatttccca tgattccttc atatttgcat
      181 atacgataca aggctgttag agagataatt ggaattaatt tgactgtaaa cacaaagata
      241 ttagtacaaa atacgtgacg tagaaagtaa taatttcttg ggtagtttgc agttttaaaa
      301 ttatgtttta aaatggacta tcatatgctt accgtaactt gaaagtattt cgatttcttg
      361 gctttatata tcttgtggaa aggacgaaac accgatgtat gaagatgatg acggttttag
      421 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg
      481 agtcggtgct ttttttctag actgcagagg gccctgcgta tgagtgcaag tgggttttag
      541 gaccaggatg aggcggggtg ggggtgccta cctgacgacc gaccccgacc cactggacaa
      601 gcacccaacc cccattcccc aaattgcgca tcccctatca gagaggggga ggggaaacag
      661 gatgcggcga ggcgcgtgcg cactgccagc ttcagcaccg cggacagtgc cttcgccccc
      721 gcctggcggc gcgcgccacc gccgcctcag cactgaaggc gcgctgacgt cactcgccgg
      781 tcccccgcaa actccccttc ccggccacct tggtcgcgtc cgcgccgccg ccggcccagc
      841 cggaccgcac cacgcgaggc gcgagatagg ggggcacggg cgcgaccatc tgcgctgcgg
      901 cgccggcgac tcagcgctgc ctcagtctgc ggtgggcagc ggaggagtcg tgtcgtgcct
      961 gagagcgcag tcgagaaggt accgccacca tgcccaagaa gaagaggaag gtgtccaatc
     1021 tcctgactgt tcaccagaac ctccctgcgc tgccagtaga tgccactagc gatgaggtca
     1081 ggaaaaatct catggatatg tttagggata gacaggcgtt ttctgaacac acctggaaaa
     1141 tgctgcttag cgtgtgccga tcctgggcag cctggtgtaa gctgaacaat cgcaaatggt
     1201 tccccgccga gccggaggac gtgcgcgatt acctgctgta tctccaggca agagggctgg
     1261 ctgtcaagac tatccagcag cacttgggcc aactgaatat gctgcatcga cgcagcgggc
     1321 tcccccggcc tagcgattca aacgcagtct cccttgttat gaggagaatt agaaaggaaa
     1381 acgtagatgc gggtgagagg gctaagcagg ctctcgcttt tgagcggact gatttcgacc
     1441 aggtcagatc cctgatggag aacagcgatc ggtgccagga catcaggaac ctcgcatttc
     1501 tgggaattgc atataacaca cttctgcgca tagctgagat cgcccggatc agagtgaaag
     1561 acatcagtcg aacggacggc ggccggatgc ttattcatat tggacgcaca aagacattgg
     1621 tcagcaccgc tggcgttgaa aaggccttgt ccctgggcgt aacgaagctg gtggaaagat
     1681 ggatctcagt gtccggcgtg gctgacgacc ctaataatta cttgttctgt cgagtgagaa
     1741 aaaacggagt cgccgcgccc tctgccacca gccaattgag tacacgggcc cttgaaggga
     1801 tctttgaggc aacccaccga ctcatatacg gagccaagga tgacagtggc cagaggtatc
     1861 tcgcctggtc aggtcattct gctagggtgg gggccgcacg agacatggcg cgggcaggag
     1921 tctccatacc agagattatg caagctggag gttggacaaa tgtgaacatc gttatgaact
     1981 atatccgcaa tcttgactct gaaaccgggg ccatggtgag actgctcgaa gatggtgact
     2041 aggaattcga tatcaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     2101 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     2161 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     2221 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     2281 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     2341 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     2401 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     2461 aatcatcgtc ctttccttgg ctgctcgcct atgttgccac ctggattctg cgcgggacgt
     2521 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     2581 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     2641 gggccgcctc cccgcatcga taccgagcgc taataaaaga tctttatttt cattagatct
     2701 gtgtgttggt tttttgtgtc acgtgcggac cgagcggccg caggaacccc tagtgatgga
     2761 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     2821 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     2881 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     2941 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3001 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     3061 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3121 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     3181 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3241 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     3301 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     3361 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     3421 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     3481 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     3541 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     3601 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     3661 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     3721 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     3781 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     3841 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     3901 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     3961 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     4021 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     4081 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     4141 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     4201 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     4261 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     4321 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     4381 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     4441 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     4501 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     4561 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     4621 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     4681 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     4741 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     4801 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     4861 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     4921 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     4981 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     5041 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     5101 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     5161 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     5221 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     5281 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     5341 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     5401 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     5461 tggccttttg ctcacatgt
//
