LOCUS       sequence-425061-        7143 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Vgat gRNA in a Cre dependent Chronos GFP vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7143)
  AUTHORS   Moffa JC, Bland IN, Tooley JR, Kalyanaraman V, Heitmeier M, Creed 
            MC, Copits BA
  TITLE     Cell-Specific Single Viral Vector CRISPR/Cas9 Editing and 
            Genetically Encoded Tool Delivery in the Central and Peripheral 
            Nervous Systems.
  JOURNAL   eNeuro. 2024 Jul 5;11(7):ENEURO.0438-23.2024. doi: 
            10.1523/ENEURO.0438-23.2024. Print 2024 Jul.
  PUBMED    38871457
REFERENCE   2  (bases 1 to 7143)
  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: 
            "10.1523/ENEURO.0438-23.2024"; journalName: "eNeuro"; date: 
            "2024-07-5- 5"; volume: "11"; issue: "7"; pages: 
            "ENEURO.0438-23.2024"
COMMENT     Sequence Label: pX552 EF1a DIO Chronos GFP_Vgat gRNA
FEATURES             Location/Qualifiers
     source          1..7143
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        271..346
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        462..1640
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          693..1631
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1588..1608
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1671..1704
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    1767..1800
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1814..2527,2528..2530))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(1817..2527,2528..2530))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1859..1880)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2208..2227
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2467..2488
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3520..3529
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3551..3584)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(3655..3688)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    3746..4222
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3945..3964)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4262..4402
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4262..4391
                     /label=AAV2 ITR
     rep_origin      4477..4932
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4564..4583)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4774..4795
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4949..4968)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5068..5090
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5128..5146)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5214..5318
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5319..5387,5388..6179)
                     /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(5537..5556)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6350..6938
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6839..6858
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   7000..7129
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ctgggacttg ttggacacgg gttttagagc tagaaatagc aagttaaaat
      301 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tttctagact
      361 gcagagggcc atcgatagct ttgcaaagat ggataaagtt ttaaacagag aggaatcttt
      421 gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt gcccgtcagt
      481 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa
      541 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc
      601 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc
      661 tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc
      721 ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt
      781 acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg
      841 cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc
      901 gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca
      961 tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg
     1021 cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc
     1081 gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg
     1141 gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta
     1201 tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat
     1261 ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc
     1321 gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat
     1381 gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg agcttttgga
     1441 gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt
     1501 gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc
     1561 tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc
     1621 ttccatttca ggtgtcgtga ggaattggat cctctagagt cgaaattccc ataacttcgt
     1681 ataaagtatc ctatacgaag ttatatcaaa ataggaagac caatgcttca ccatcgaccc
     1741 gaattgccaa gcatcaccat cgacccataa cttcgtataa tgtatgctat acgaagttat
     1801 actagctagc tcattacttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
     1861 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact
     1921 gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     1981 gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt
     2041 tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt
     2101 tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct
     2161 cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc
     2221 cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga
     2281 agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca
     2341 gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcaa atgaacttca
     2401 gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc
     2461 cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc
     2521 ccttgctcac tgctactacc ggtgccgcca ctcctccctc ctcctcctcg tcgacgaagg
     2581 tctcgatctc catgttctcg ccggccacgt tgatggtggt tgtcttccgg atgtcgccgt
     2641 ggatcaggat gtgctcgtgg atcttcactc tcaggtggtg gcccaggaag ccccaagtgt
     2701 tcttgctgat cagatccagg atcaggtggc cgattccgct ggtgtaaggt gtgatcaggc
     2761 ccagtccctc gtgtccggcg atgaacatca cggggaacat cagccaagag cagaagaaga
     2821 cgtaggccat gatcttgcaa atcttccggc agacgccttt gggcagggtg tggtagctct
     2881 cgatatacac cttggcgatc tggaagaagc aagtcacgcc gtagaacagg ccgatcatga
     2941 agaacaggat cttcaggggg ccctttgtaa aggcggctgt gatcccccac acgatgttgc
     3001 cgatgtcggt caccaggatg gtcatggtcc gcttgctgta ctcttcgtgc agtccggtca
     3061 ggttgctcag gtggatcagg atcacggggc aagtcaggag ccacatgctg taccgcagcc
     3121 aaatcacggc tcctccgttg gtctggtaca ctgtggctgg gctgtcgacc tcgtggaaca
     3181 gctcgatgaa gcacttgacg agctcgatca cgcagacgta gacctcctcc cagccggttg
     3241 tggccttgaa gctgtgccaa ccgtagaaga tgagctgcac gatagccacg atcacggtga
     3301 accactggag tcccactgcg atcttgtgct gcagctcggt tccgtggttg atgtggggga
     3361 agcaaccgtg atcggctccg gctgttgtgg cgttagaggt ttctccgtga gcgtctgctg
     3421 ctggtgtcac cactgctgcg gcgctcagca ggcctctaat tgtggcttcg gcgctaggca
     3481 cggctgagat aaaggcgtgg gtcattgtgg cggctgtttc catggtggcg gcgcggccta
     3541 ggtacctagt ataacttcgt ataggatact ttatacgaag ttatcattgg gattcttcct
     3601 attttgatcc aagcatcacc atcgaccctc tagtccagat ctcaccatcg acccataact
     3661 tcgtatagca tacattatac gaagttatgt ccctcgaaga ggttcgcgaa gcttatcgat
     3721 accgagcgct gctcgagaga tctacgggtg gcatccctgt gacccctccc cagtgcctct
     3781 cctggccctg gaagttgcca ctccagtgcc caccagcctt gtcctaataa aattaagttg
     3841 catcattttg tctgactagg tgtccttcta taatattatg gggtggaggg gggtggtatg
     3901 gagcaagggg caagttggga agacaacctg tagggcctgc ggggtctatt gggaaccaag
     3961 ctggagtgca gtggcacaat cttggctcac tgcaatctcc gcctcctggg ttcaagcgat
     4021 tctcctgcct cagcctcccg agttgttggg attccaggca tgcatgacca ggctcagcta
     4081 atttttgttt ttttggtaga gacggggttt caccatattg gccaggctgg tctccaactc
     4141 ctaatctcag gtgatctacc caccttggcc tcccaaattg ctgggattac aggcgtgaac
     4201 cactgctccc ttccctgtcc ttctgatttt gtaggtaacc acgtgcggac cgagcggccg
     4261 caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag
     4321 gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag
     4381 cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac gcatctgtgc
     4441 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
     4501 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccttagcgc
     4561 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     4621 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     4681 aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     4741 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     4801 cactcaactc tatctcgggc tattcttttg atttataagg gattttgccg atttcggtct
     4861 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     4921 cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     4981 agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat
     5041 ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt
     5101 catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg
     5161 tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa
     5221 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     5281 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     5341 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     5401 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     5461 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     5521 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     5581 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     5641 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     5701 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     5761 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     5821 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     5881 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     5941 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     6001 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     6061 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     6121 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     6181 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     6241 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     6301 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     6361 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     6421 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     6481 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     6541 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     6601 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     6661 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     6721 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     6781 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     6841 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     6901 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     6961 tacggttcct ggccttttgc tggccttttg ctcacatgtc ctgcaggcag ctgcgcgctc
     7021 gctcgctcac tgaggccgcc cgggcgtcgg gcgacctttg gtcgcccggc ctcagtgagc
     7081 gagcgagcgc gcagagaggg agtggccaac tccatcacta ggggttcctg cggccgcacg
     7141 cgt
//
