LOCUS       sequence-231549-        7219 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Chronos with red nucleus-targeted fluorescence for inhibitory 
            neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7219)
  TITLE     Chronos with red nucleus-targeted fluorescence for excitatory 
            neurons
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7219)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pAAV-Dlx5/6-Chronos-eGFP-p2a-nls-mScarlet
FEATURES             Location/Qualifiers
     source          1..7219
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     intron          860..992
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     975..994
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     CDS             join(2022..2024,2025..2735)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2022..2024,2025..2735)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2064..2085)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2325..2344)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2672..2693
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2745..2801
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2805..2825
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2959..2977)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3227..3246
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    3565..4153
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3618..3638)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4036..4047)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4322..4403
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4359..4378)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4413..4432
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4482..4622
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4482..4611
                     /label=AAV2 ITR
     rep_origin      4697..5152
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4784..4803)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4994..5015
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5169..5188)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5288..5310
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5348..5366)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5434..5538
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5539..5607,5608..6399)
                     /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(5757..5776)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6570..7158
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7059..7078
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtttaat taagaattct atacactcac
      181 agtggtttgg catatatttg gtgaaatttt ttaaggaaaa attagtgttg gtttcgatat
      241 atggtagctt tttctctaac ataatttgaa taattcagca aagccctact accagctgta
      301 cttctgcagc ctcttccatt ctttccagca ttataatttt ggttaatttt caattttagg
      361 tcctacgtct ctgcaatttg tgtatgaata acagaataat ttccctcttt tgtttcgcct
      421 ttcctgttcc tgaatctaaa taaagatggc tttttagtat taaaagtgga agaaaattac
      481 aggtaattat ctttgacggt aaaaacgctg taatcagcgg gctacatgaa aaattactct
      541 aattatggct gcatttaaga gaatggaaaa aaaccttctt gtggataaaa accttaaatt
      601 gtccccaatg tctgcttcaa attggatggc actgcagctg gaggctttgt tcagaattga
      661 tcctggggag ctacgaaccc aaagtttcac agtaggaagc ttgatatcga attcctgcag
      721 cccgggctgg gcataaaagt cagggcagag ccatctattg cttacatttg cttctagcct
      781 gcaggtcgag gagcgcagcc ttccagaagc agagcgcggc gccttaagct gcagaagttg
      841 gtcgtgaggc actgggcagg taagtatcaa ggttacaaga caggtttaag gagaccaata
      901 gaaactgggc ttgtcgagac agagaagact cttgcgtttc tgataggcac ctattggtct
      961 tactgacatc cactttgcct ttctctccac aggtgtccac tcccagttca attacagctc
     1021 ttaagaaact agtgccatgg gccacaatga cccacgcctt tatctcagcc gtgcctagcg
     1081 ccgaagccac aattagaggc ctgctgagcg ccgcagcagt ggtgacacca gcagcagacg
     1141 ctcacggaga aacctctaac gccacaacag ccggagccga tcacggttgc ttcccccaca
     1201 tcaaccacgg aaccgagctg cagcacaaga tcgcagtggg actccagtgg ttcaccgtga
     1261 tcgtggctat cgtgcagctc atcttctacg gttggcacag cttcaaggcc acaaccggct
     1321 gggaggaggt ctacgtctgc gtgatcgagc tcgtcaagtg cttcatcgag ctgttccacg
     1381 aggtcgacag cccagccaca gtgtaccaga ccaacggagg agccgtgatt tggctgcggt
     1441 acagcatgtg gctcctgact tgccccgtga tcctgatcca cctgagcaac ctgaccggac
     1501 tgcacgaaga gtacagcaag cggaccatga ccatcctggt gaccgacatc ggcaacatcg
     1561 tgtgggggat cacagccgcc tttacaaagg gccccctgaa gatcctgttc ttcatgatcg
     1621 gcctgttcta cggcgtgact tgcttcttcc agatcgccaa ggtgtatatc gagagctacc
     1681 acaccctgcc caaaggcgtc tgccggaaga tttgcaagat catggcctac gtcttcttct
     1741 gctcttggct gatgttcccc gtgatgttca tcgccggaca cgagggactg ggcctgatca
     1801 caccttacac cagcggaatc ggccacctga tcctggatct gatcagcaag aacacttggg
     1861 gcttcctggg ccaccacctg agagtgaaga tccacgagca catcctgatc cacggcgaca
     1921 tccggaagac aaccaccatc aacgtggccg gcgagaacat ggagatcgag accttcgtcg
     1981 acgaggagga ggagggagga gtggcggcac cggtagtagc agtgagcaag ggcgaggagc
     2041 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     2101 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca
     2161 tttgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     2221 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2281 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca
     2341 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2401 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca
     2461 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga
     2521 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     2581 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc
     2641 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2701 ccgggatcac tctcggcatg gacgagctgt acaagggatc cggagccacg aacttctctc
     2761 tgttaaagca agcaggagac gtggaagaaa accccggtcc tgacccaaaa aagaagcgga
     2821 aggtcatggt gagcaagggc gaggcagtga tcaaggagtt catgcggttc aaggtgcaca
     2881 tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag ggccgcccct
     2941 acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg cccttctcct
     3001 gggacatcct gtcccctcag ttcatgtacg gctccagggc cttcaccaag caccccgccg
     3061 acatccccga ctactataag cagtccttcc ccgagggctt caagtgggag cgcgtgatga
     3121 acttcgagga cggcggcgcc gtgaccgtga cccaggacac ctccctggag gacggcaccc
     3181 tgatctacaa ggtgaagctc cgcggcacca acttccctcc tgacggcccc gtaatgcaga
     3241 agaagacaat gggctgggaa gcgtccaccg agcggttgta ccccgaggac ggcgtgctga
     3301 agggcgacat taagatggcc ctgcgcctga aggacggcgg acgctacctg gcggacttca
     3361 agaccaccta caaggccaag aagcccgtgc agatgcccgg cgcctacaac gtcgaccgca
     3421 agttggacat cacctcccac aacgaggact acaccgtggt ggaacagtac gaacgctccg
     3481 agggccgcca ctccaccggc gccagagcag caccttaata aggcgcgcca cccctgcagg
     3541 gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg tgaaagattg
     3601 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     3661 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     3721 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     3781 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     3841 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     3901 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     3961 tcatcgtcct ttccttggct gctcgcctat gttgccacct ggattctgcg cgggacgtcc
     4021 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     4081 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     4141 gccgcctccc cgcatcgata ccgagcgctg ctcgagagat ctgtgatagc ggccatcaag
     4201 ctggccgcga ctctagatca taatcagcca taccacattt gtagaggttt tacttgcttt
     4261 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     4321 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     4381 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     4441 agcttatcga taccgcatgc acgtgcggac cgagcggccg caggaacccc tagtgatgga
     4501 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     4561 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     4621 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     4681 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4741 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4801 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4861 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4921 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4981 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc
     5041 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5101 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     5161 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     5221 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5281 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     5341 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     5401 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5461 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5521 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5581 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5641 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5701 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5761 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5821 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5881 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5941 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     6001 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     6061 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     6121 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6181 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6241 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6301 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6361 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6421 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6481 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6541 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6601 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6661 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     6721 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6781 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6841 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     6901 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     6961 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     7021 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     7081 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     7141 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     7201 tggccttttg ctcacatgt
//
