LOCUS       sequence-394121-        7140 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Chronos-eGFP in a Cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7140)
  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 7140)
  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-eGFP(with gRNA scaffold)
FEATURES             Location/Qualifiers
     source          1..7140
                     /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        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        459..1637
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          690..1628
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1585..1605
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    1668..1701
                     /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    1764..1797
                     /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(1811..2524,2525..2527))
                     /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(1814..2524,2525..2527))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1856..1877)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2205..2224
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2464..2485
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3517..3526
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3548..3581)
                     /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(3652..3685)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    3743..4219
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3942..3961)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4259..4399
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4259..4388
                     /label=AAV2 ITR
     rep_origin      4474..4929
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4561..4580)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4771..4792
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4946..4965)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5065..5087
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5125..5143)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5211..5315
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5316..5384,5385..6176)
                     /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(5534..5553)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6347..6935
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6836..6855
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6997..7126
                     /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 gaagagcgag ctcttctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt ctagactgca
      361 gagggccatc gatagctttg caaagatgga taaagtttta aacagagagg aatctttgca
      421 gctaatggac cttctaggtc ttgaaaggag tgggaattgg ctccggtgcc cgtcagtggg
      481 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg
      541 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
      601 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
      661 ttcgcaacgg gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg
      721 gcctctttac gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctgcagtacg
      781 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
      841 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
      901 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
      961 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
     1021 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
     1081 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
     1141 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg
     1201 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
     1261 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
     1321 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
     1381 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
     1441 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     1501 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     1561 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     1621 catttcaggt gtcgtgagga attggatcct ctagagtcga aattcccata acttcgtata
     1681 aagtatccta tacgaagtta tatcaaaata ggaagaccaa tgcttcacca tcgacccgaa
     1741 ttgccaagca tcaccatcga cccataactt cgtataatgt atgctatacg aagttatact
     1801 agctagctca ttacttgtac agctcgtcca tgccgagagt gatcccggcg gcggtcacga
     1861 actccagcag gaccatgtga tcgcgcttct cgttggggtc tttgctcagg gcggactggg
     1921 tgctcaggta gtggttgtcg ggcagcagca cggggccgtc gccgatgggg gtgttctgct
     1981 ggtagtggtc ggcgagctgc acgctgccgt cctcgatgtt gtggcggatc ttgaagttca
     2041 ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt
     2101 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
     2161 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
     2221 cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
     2281 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg
     2341 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcaaatg aacttcaggg
     2401 tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt
     2461 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
     2521 tgctcactgc tactaccggt gccgccactc ctccctcctc ctcctcgtcg acgaaggtct
     2581 cgatctccat gttctcgccg gccacgttga tggtggttgt cttccggatg tcgccgtgga
     2641 tcaggatgtg ctcgtggatc ttcactctca ggtggtggcc caggaagccc caagtgttct
     2701 tgctgatcag atccaggatc aggtggccga ttccgctggt gtaaggtgtg atcaggccca
     2761 gtccctcgtg tccggcgatg aacatcacgg ggaacatcag ccaagagcag aagaagacgt
     2821 aggccatgat cttgcaaatc ttccggcaga cgcctttggg cagggtgtgg tagctctcga
     2881 tatacacctt ggcgatctgg aagaagcaag tcacgccgta gaacaggccg atcatgaaga
     2941 acaggatctt cagggggccc tttgtaaagg cggctgtgat cccccacacg atgttgccga
     3001 tgtcggtcac caggatggtc atggtccgct tgctgtactc ttcgtgcagt ccggtcaggt
     3061 tgctcaggtg gatcaggatc acggggcaag tcaggagcca catgctgtac cgcagccaaa
     3121 tcacggctcc tccgttggtc tggtacactg tggctgggct gtcgacctcg tggaacagct
     3181 cgatgaagca cttgacgagc tcgatcacgc agacgtagac ctcctcccag ccggttgtgg
     3241 ccttgaagct gtgccaaccg tagaagatga gctgcacgat agccacgatc acggtgaacc
     3301 actggagtcc cactgcgatc ttgtgctgca gctcggttcc gtggttgatg tgggggaagc
     3361 aaccgtgatc ggctccggct gttgtggcgt tagaggtttc tccgtgagcg tctgctgctg
     3421 gtgtcaccac tgctgcggcg ctcagcaggc ctctaattgt ggcttcggcg ctaggcacgg
     3481 ctgagataaa ggcgtgggtc attgtggcgg ctgtttccat ggtggcggcg cggcctaggt
     3541 acctagtata acttcgtata ggatacttta tacgaagtta tcattgggat tcttcctatt
     3601 ttgatccaag catcaccatc gaccctctag tccagatctc accatcgacc cataacttcg
     3661 tatagcatac attatacgaa gttatgtccc tcgaagaggt tcgcgaagct tatcgatacc
     3721 gagcgctgct cgagagatct acgggtggca tccctgtgac ccctccccag tgcctctcct
     3781 ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat taagttgcat
     3841 cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg tggtatggag
     3901 caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg aaccaagctg
     3961 gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc aagcgattct
     4021 cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc tcagctaatt
     4081 tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct ccaactccta
     4141 atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg cgtgaaccac
     4201 tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga gcggccgcag
     4261 gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc
     4321 gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga
     4381 gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     4441 atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg
     4501 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg
     4561 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc
     4621 taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
     4681 aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc
     4741 ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac
     4801 tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt
     4861 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt
     4921 ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc
     4981 cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg
     5041 cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat
     5101 caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca
     5161 tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
     5221 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct
     5281 gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg
     5341 cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg
     5401 tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc
     5461 tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca
     5521 cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac
     5581 tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa
     5641 agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg
     5701 ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt
     5761 ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg
     5821 aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc
     5881 gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga
     5941 tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta
     6001 ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc
     6061 cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg
     6121 atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt
     6181 cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa
     6241 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt
     6301 cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt
     6361 ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt
     6421 tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga
     6481 taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag
     6541 caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata
     6601 agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg
     6661 gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga
     6721 gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca
     6781 ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa
     6841 acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt
     6901 tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac
     6961 ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg cgcgctcgct
     7021 cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc agtgagcgag
     7081 cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg ccgcacgcgt
//
