LOCUS       sequence-405512-        5477 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone plasmid for cloning HiUGE GS-gRNA (with hSyn-Cre).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5477)
  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 5477)
  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) backbone
FEATURES             Location/Qualifiers
     source          1..5477
                     /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        412..487
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        521..968
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     CDS             991..1011
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     misc_feature    2065..2653
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2118..2138)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2536..2547)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     repeat_region   2740..2880
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2740..2869
                     /label=AAV2 ITR
     rep_origin      2955..3410
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3042..3061)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3252..3273
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3427..3446)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3546..3568
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3606..3624)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3692..3796
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3797..3865,3866..4657)
                     /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(4015..4034)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4828..5416
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5317..5336
                     /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 accggaagag cgagctcttc tgttttagag
      421 ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
      481 tcggtgcttt ttttctagac tgcagagggc cctgcgtatg agtgcaagtg ggttttagga
      541 ccaggatgag gcggggtggg ggtgcctacc tgacgaccga ccccgaccca ctggacaagc
      601 acccaacccc cattccccaa attgcgcatc ccctatcaga gagggggagg ggaaacagga
      661 tgcggcgagg cgcgtgcgca ctgccagctt cagcaccgcg gacagtgcct tcgcccccgc
      721 ctggcggcgc gcgccaccgc cgcctcagca ctgaaggcgc gctgacgtca ctcgccggtc
      781 ccccgcaaac tccccttccc ggccaccttg gtcgcgtccg cgccgccgcc ggcccagccg
      841 gaccgcacca cgcgaggcgc gagatagggg ggcacgggcg cgaccatctg cgctgcggcg
      901 ccggcgactc agcgctgcct cagtctgcgg tgggcagcgg aggagtcgtg tcgtgcctga
      961 gagcgcagtc gagaaggtac cgccaccatg cccaagaaga agaggaaggt gtccaatctc
     1021 ctgactgttc accagaacct ccctgcgctg ccagtagatg ccactagcga tgaggtcagg
     1081 aaaaatctca tggatatgtt tagggataga caggcgtttt ctgaacacac ctggaaaatg
     1141 ctgcttagcg tgtgccgatc ctgggcagcc tggtgtaagc tgaacaatcg caaatggttc
     1201 cccgccgagc cggaggacgt gcgcgattac ctgctgtatc tccaggcaag agggctggct
     1261 gtcaagacta tccagcagca cttgggccaa ctgaatatgc tgcatcgacg cagcgggctc
     1321 ccccggccta gcgattcaaa cgcagtctcc cttgttatga ggagaattag aaaggaaaac
     1381 gtagatgcgg gtgagagggc taagcaggct ctcgcttttg agcggactga tttcgaccag
     1441 gtcagatccc tgatggagaa cagcgatcgg tgccaggaca tcaggaacct cgcatttctg
     1501 ggaattgcat ataacacact tctgcgcata gctgagatcg cccggatcag agtgaaagac
     1561 atcagtcgaa cggacggcgg ccggatgctt attcatattg gacgcacaaa gacattggtc
     1621 agcaccgctg gcgttgaaaa ggccttgtcc ctgggcgtaa cgaagctggt ggaaagatgg
     1681 atctcagtgt ccggcgtggc tgacgaccct aataattact tgttctgtcg agtgagaaaa
     1741 aacggagtcg ccgcgccctc tgccaccagc caattgagta cacgggccct tgaagggatc
     1801 tttgaggcaa cccaccgact catatacgga gccaaggatg acagtggcca gaggtatctc
     1861 gcctggtcag gtcattctgc tagggtgggg gccgcacgag acatggcgcg ggcaggagtc
     1921 tccataccag agattatgca agctggaggt tggacaaatg tgaacatcgt tatgaactat
     1981 atccgcaatc ttgactctga aaccggggcc atggtgagac tgctcgaaga tggtgactag
     2041 gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     2101 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     2161 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     2221 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     2281 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     2341 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     2401 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     2461 tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg cgggacgtcc
     2521 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     2581 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     2641 gccgcctccc cgcatcgata ccgagcgcta ataaaagatc tttattttca ttagatctgt
     2701 gtgttggttt tttgtgtcac gtgcggaccg agcggccgca ggaaccccta gtgatggagt
     2761 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     2821 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     2881 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatacgtc
     2941 aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3001 gcgcagcgtg accgctacac ttgccagcgc cttagcgccc gctcctttcg ctttcttccc
     3061 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3121 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt tgggtgatgg
     3181 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3241 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaactcta tctcgggcta
     3301 ttcttttgat ttataaggga ttttgccgat ttcggtctat tggttaaaaa atgagctgat
     3361 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt tatggtgcac
     3421 tctcagtaca atctgctctg atgccgcata gttaagccag ccccgacacc cgccaacacc
     3481 cgctgacgcg ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac
     3541 cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg
     3601 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     3661 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     3721 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     3781 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     3841 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     3901 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     3961 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     4021 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     4081 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     4141 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     4201 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     4261 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     4321 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga
     4381 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     4441 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     4501 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     4561 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     4621 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     4681 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     4741 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     4801 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     4861 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     4921 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct
     4981 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     5041 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     5101 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     5161 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     5221 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     5281 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     5341 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     5401 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     5461 gccttttgct cacatgt
//
