LOCUS       sequence-235975-        7208 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 7208)
  TITLE     Chronos with red nucleus-targeted fluorescence for excitatory 
            neurons
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7208)
  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            "Unpublished"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..7208
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1030..1032,1033..1743)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1030..1032,1033..1743)
                     /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(1072..1093)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1333..1352)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1680..1701
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1753..1809
                     /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             1813..1833
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(1967..1985)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2235..2254
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    2573..3161
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2626..2646)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3044..3055)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3330..3411
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3367..3386)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3421..3440
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3490..3630
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3490..3619
                     /label=AAV2 ITR
     rep_origin      3705..4160
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3792..3811)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4002..4023
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4177..4196)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4296..4318
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4356..4374)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4442..4546
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4547..4615,4616..5407)
                     /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(4765..4784)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5578..6166
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6067..6086
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   6228..6357
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     intron          7076..7208
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     join(7191..7208,1..2)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
ORIGIN
        1 gtgtccactc ccagttcaat tacagctctt aagaaactag tgccatgggc cacaatgacc
       61 cacgccttta tctcagccgt gcctagcgcc gaagccacaa ttagaggcct gctgagcgcc
      121 gcagcagtgg tgacaccagc agcagacgct cacggagaaa cctctaacgc cacaacagcc
      181 ggagccgatc acggttgctt cccccacatc aaccacggaa ccgagctgca gcacaagatc
      241 gcagtgggac tccagtggtt caccgtgatc gtggctatcg tgcagctcat cttctacggt
      301 tggcacagct tcaaggccac aaccggctgg gaggaggtct acgtctgcgt gatcgagctc
      361 gtcaagtgct tcatcgagct gttccacgag gtcgacagcc cagccacagt gtaccagacc
      421 aacggaggag ccgtgatttg gctgcggtac agcatgtggc tcctgacttg ccccgtgatc
      481 ctgatccacc tgagcaacct gaccggactg cacgaagagt acagcaagcg gaccatgacc
      541 atcctggtga ccgacatcgg caacatcgtg tgggggatca cagccgcctt tacaaagggc
      601 cccctgaaga tcctgttctt catgatcggc ctgttctacg gcgtgacttg cttcttccag
      661 atcgccaagg tgtatatcga gagctaccac accctgccca aaggcgtctg ccggaagatt
      721 tgcaagatca tggcctacgt cttcttctgc tcttggctga tgttccccgt gatgttcatc
      781 gccggacacg agggactggg cctgatcaca ccttacacca gcggaatcgg ccacctgatc
      841 ctggatctga tcagcaagaa cacttggggc ttcctgggcc accacctgag agtgaagatc
      901 cacgagcaca tcctgatcca cggcgacatc cggaagacaa ccaccatcaa cgtggccggc
      961 gagaacatgg agatcgagac cttcgtcgac gaggaggagg agggaggagt ggcggcaccg
     1021 gtagtagcag tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     1081 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     1141 acctacggca agctgaccct gaagttcatt tgcaccaccg gcaagctgcc cgtgccctgg
     1201 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     1261 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     1321 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     1381 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     1441 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     1501 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     1561 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     1621 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1681 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1741 aagggatccg gagccacgaa cttctctctg ttaaagcaag caggagacgt ggaagaaaac
     1801 cccggtcctg acccaaaaaa gaagcggaag gtcatggtga gcaagggcga ggcagtgatc
     1861 aaggagttca tgcggttcaa ggtgcacatg gagggctcca tgaacggcca cgagttcgag
     1921 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg
     1981 accaagggtg gccccctgcc cttctcctgg gacatcctgt cccctcagtt catgtacggc
     2041 tccagggcct tcaccaagca ccccgccgac atccccgact actataagca gtccttcccc
     2101 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgccgt gaccgtgacc
     2161 caggacacct ccctggagga cggcaccctg atctacaagg tgaagctccg cggcaccaac
     2221 ttccctcctg acggccccgt aatgcagaag aagacaatgg gctgggaagc gtccaccgag
     2281 cggttgtacc ccgaggacgg cgtgctgaag ggcgacatta agatggccct gcgcctgaag
     2341 gacggcggac gctacctggc ggacttcaag accacctaca aggccaagaa gcccgtgcag
     2401 atgcccggcg cctacaacgt cgaccgcaag ttggacatca cctcccacaa cgaggactac
     2461 accgtggtgg aacagtacga acgctccgag ggccgccact ccaccggcgc cagagcagca
     2521 ccttaataag gcgcgccacc cctgcaggga attcgatatc aagcttatcg ataatcaacc
     2581 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     2641 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     2701 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     2761 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     2821 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     2881 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     2941 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctatgt
     3001 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     3061 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     3121 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gagcgctgct
     3181 cgagagatct gtgatagcgg ccatcaagct ggccgcgact ctagatcata atcagccata
     3241 ccacatttgt agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga
     3301 aacataaaat gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca
     3361 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     3421 gtggtttgtc caaactcatc aatgtatcag cttatcgata ccgcatgcac gtgcggaccg
     3481 agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc
     3541 tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag
     3601 tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt ctccttacgc
     3661 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     3721 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3781 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3841 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3901 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3961 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4021 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     4081 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4141 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     4201 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     4261 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     4321 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     4381 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     4441 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     4501 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     4561 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     4621 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     4681 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     4741 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     4801 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     4861 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     4921 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     4981 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5041 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5101 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5161 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     5221 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     5281 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     5341 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     5401 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     5461 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     5521 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     5581 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     5641 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     5701 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     5761 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     5821 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     5881 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     5941 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     6001 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     6061 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     6121 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     6181 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtcct gcaggcagct
     6241 gcgcgctcgc tcgctcactg aggccgcccg ggcgtcgggc gacctttggt cgcccggcct
     6301 cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg ggttcctgcg
     6361 gccgcacgcg tttaattaag aattctatac actcacagtg gtttggcata tatttggtga
     6421 aattttttaa ggaaaaatta gtgttggttt cgatatatgg tagctttttc tctaacataa
     6481 tttgaataat tcagcaaagc cctactacca gctgtacttc tgcagcctct tccattcttt
     6541 ccagcattat aattttggtt aattttcaat tttaggtcct acgtctctgc aatttgtgta
     6601 tgaataacag aataatttcc ctcttttgtt tcgcctttcc tgttcctgaa tctaaataaa
     6661 gatggctttt tagtattaaa agtggaagaa aattacaggt aattatcttt gacggtaaaa
     6721 acgctgtaat cagcgggcta catgaaaaat tactctaatt atggctgcat ttaagagaat
     6781 ggaaaaaaac cttcttgtgg ataaaaacct taaattgtcc ccaatgtctg cttcaaattg
     6841 gatggcactg cagctggagg ctttgttcag aattgatcct ggggagctac gaacccaaag
     6901 tttcacagta ggaagcttga tatcgaattc ctgcagcccg ggctgggcat aaaagtcagg
     6961 gcagagccat ctattgctta catttgcttc tagcctgcag gtcgaggagc gcagccttcc
     7021 agaagcagag cgcggcgcct taagctgcag aagttggtcg tgaggcactg ggcaggtaag
     7081 tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt cgagacagag
     7141 aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact ttgcctttct
     7201 ctccacag
//
