LOCUS       sequence-308124-        6249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Chronos with red nucleus-targeted fluorescence for excitatory 
            neurons.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6249)
  TITLE     Chronos with red nucleus-targeted fluorescence for excitatory 
            neurons
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6249)
  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            "Unpublished"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: peAAV-CKII(0.4)-Chronos_eGFP_GP2A_nls_mScarlet
FEATURES             Location/Qualifiers
     source          1..6249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(8..29)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(269..288)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     616..637
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             689..745
                     /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             749..769
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             770..1465
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(903..921)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1171..1190
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    1487..1890
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     polyA_signal    1897..1945
                     /note="synthetic polyadenylation signal"
     repeat_region   1975..2115
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1975..2104
                     /label=AAV2 ITR
     rep_origin      2190..2645
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2277..2296)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2487..2508
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2662..2681)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2781..2803
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2841..2859)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2927..3031
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3032..3100,3101..3892)
                     /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(3250..3269)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4063..4651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4552..4571
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   4713..4842
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     CDS             join(6215..6217,6218..6249,1..679)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6215..6217,6218..6249,1..679)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
ORIGIN
        1 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
       61 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatttgca ccaccggcaa
      121 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
      181 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
      241 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
      301 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
      361 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat
      421 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga
      481 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
      541 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
      601 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
      661 catggacgag ctgtacaagg gatccggagc cacgaacttc tctctgttaa agcaagcagg
      721 agacgtggaa gaaaaccccg gtcctgaccc aaaaaagaag cggaaggtca tggtgagcaa
      781 gggcgaggca gtgatcaagg agttcatgcg gttcaaggtg cacatggagg gctccatgaa
      841 cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gcacccagac
      901 cgccaagctg aaggtgacca agggtggccc cctgcccttc tcctgggaca tcctgtcccc
      961 tcagttcatg tacggctcca gggccttcac caagcacccc gccgacatcc ccgactacta
     1021 taagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg
     1081 cgccgtgacc gtgacccagg acacctccct ggaggacggc accctgatct acaaggtgaa
     1141 gctccgcggc accaacttcc ctcctgacgg ccccgtaatg cagaagaaga caatgggctg
     1201 ggaagcgtcc accgagcggt tgtaccccga ggacggcgtg ctgaagggcg acattaagat
     1261 ggccctgcgc ctgaaggacg gcggacgcta cctggcggac ttcaagacca cctacaaggc
     1321 caagaagccc gtgcagatgc ccggcgccta caacgtcgac cgcaagttgg acatcacctc
     1381 ccacaacgag gactacaccg tggtggaaca gtacgaacgc tccgagggcc gccactccac
     1441 cggcggcatg gacgagctgt acaagtagga attcgatatc aagcttaatc aacctctgga
     1501 ttacaaaatt tgtgaaagat tgactgatat tcttaactat gttgctcctt ttacgctgtg
     1561 tggatatgct gctttaatgc ctctgtatca tgctattgct tcccgtatgg ctttcgtttt
     1621 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtccg
     1681 tcaacgtggc gtggtgtgct ctgtgtttgc tgacgcaacc cccactggct ggggcattgc
     1741 caccacctgt caactccttt ctgggacttt cgctttcccc ctcccgatcg ccacggcaga
     1801 actcatcgcc gcctgccttg cccgctgctg gacaggggct aggttgctgg gcactgataa
     1861 ttccgtggtg ttgtcgggga agctgacgtc agcgctaata aaatcgctat ccatcgaaga
     1921 tggatgtgtg ttggtttttt gtgtgggtaa cccacgtgcg gaccgagcgg ccgcaggaac
     1981 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     2041 gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc
     2101 gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     2161 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     2221 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccttag cgcccgctcc
     2281 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2341 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2401 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2461 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2521 ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt
     2581 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     2641 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     2701 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     2761 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     2821 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     2881 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     2941 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3001 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3061 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3121 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     3181 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     3241 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     3301 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     3361 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     3421 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     3481 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     3541 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     3601 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     3661 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     3721 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     3781 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     3841 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     3901 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     3961 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4021 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4081 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4141 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     4201 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     4261 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     4321 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     4381 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     4441 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     4501 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     4561 ctggtatctt tatagtcctg tcgggttttg ccacctctga cttgagcgtc gatttttgtg
     4621 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     4681 cctggccttt tgctggcctt ttgctcacat gtcctgcagg cagctgcgcg ctcgctcgct
     4741 cactgaggcc gcccgggcgt cgggcgacct ttggtcgccc ggcctcagtg agcgagcgag
     4801 cgcgcagaga gggagtggcc aactccatca ctaggggttc ctgcggccgc acgcgtcttg
     4861 tggactaagt ttgttcgcat ccccttctcc aaccccctca gtacatcacc ctgggggaac
     4921 agggtccact tgctcctggg cccacacagt cctgcagtat tgtgtatata aggccagggc
     4981 aaagaggagc aggttttaaa gtgaaaggca ggcaggtgtt ggggaggcag ttaccggggc
     5041 aacgggaaca gggcgtttcg gaggtggttg ccatggggac ctggatgctg acgaaggctc
     5101 gcgaggctgt gagcagccac agtgccctgc tcagaagccc caagctcgtc agtcaagccg
     5161 gttctccgtt tgcactcagg agcacgggca ggcgagtggc ccctagttct gggggcagct
     5221 ctagagcggt accgccacaa tgacccacgc ctttatctca gccgtgccta gcgccgaagc
     5281 cacaattaga ggcctgctga gcgccgcagc agtggtgaca ccagcagcag acgctcacgg
     5341 agaaacctct aacgccacaa cagccggagc cgatcacggt tgcttccccc acatcaacca
     5401 cggaaccgag ctgcagcaca agatcgcagt gggactccag tggttcaccg tgatcgtggc
     5461 tatcgtgcag ctcatcttct acggttggca cagcttcaag gccacaaccg gctgggagga
     5521 ggtctacgtc tgcgtgatcg agctcgtcaa gtgcttcatc gagctgttcc acgaggtcga
     5581 cagcccagcc acagtgtacc agaccaacgg aggagccgtg atttggctgc ggtacagcat
     5641 gtggctcctg acttgccccg tgatcctgat ccacctgagc aacctgaccg gactgcacga
     5701 agagtacagc aagcggacca tgaccatcct ggtgaccgac atcggcaaca tcgtgtgggg
     5761 gatcacagcc gcctttacaa agggccccct gaagatcctg ttcttcatga tcggcctgtt
     5821 ctacggcgtg acttgcttct tccagatcgc caaggtgtat atcgagagct accacaccct
     5881 gcccaaaggc gtctgccgga agatttgcaa gatcatggcc tacgtcttct tctgctcttg
     5941 gctgatgttc cccgtgatgt tcatcgccgg acacgaggga ctgggcctga tcacacctta
     6001 caccagcgga atcggccacc tgatcctgga tctgatcagc aagaacactt ggggcttcct
     6061 gggccaccac ctgagagtga agatccacga gcacatcctg atccacggcg acatccggaa
     6121 gacaaccacc atcaacgtgg ccggcgagaa catggagatc gagaccttcg tcgacgagga
     6181 ggaggaggga ggagtggcgg caccggtagt agcagtgagc aagggcgagg agctgttcac
     6241 cggggtggt
//
