LOCUS       sequence-91563-e        6827 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression vector for Chronos-GFP in a floxed/reversed, 
            Cre-dependent manner (Cre turns gene on).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6827)
  AUTHORS   Klapoetke NC, Murata Y, Kim SS, Pulver SR, Birdsey-Benson A, Cho YK,
            Morimoto TK, Chuong AS, Carpenter EJ, Tian Z, Wang J, Xie Y, Yan Z, 
            Zhang Y, Chow BY, Surek B, Melkonian M, Jayaraman V, 
            Constantine-Paton M, Wong GK, Boyden ES
  TITLE     Independent optical excitation of distinct neural populations.
  JOURNAL   Nat Methods. 2014 Mar;11(3):338-46. doi: 10.1038/nmeth.2836. Epub 
            2014 Feb 9.
  PUBMED    24509633
REFERENCE   2  (bases 1 to 6827)
  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: 
            "10.1038/nmeth.2836"; journalName: "Nat Methods"; date: "2014-03"; 
            volume: "11"; issue: "3"; pages: "338-46"
COMMENT     Sequence Label: pCAG-FLEX-rc[Chronos-GFP]
FEATURES             Location/Qualifiers
     source          1..6827
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..660
                     /label=chicken beta-actin promoter
     intron          661..1674
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1598..1620
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1682..1701
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1731..1764
                     /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    1827..1860
                     /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(1874..2587,2588..2590))
                     /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(1877..2587,2588..2590))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1919..1940)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2268..2287
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2527..2548
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      3580..3589
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(3611..3644)
                     /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(3715..3748)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3851..3870)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3916..3971
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3970..3989)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4330..4346)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4330..4346)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4343..4365)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4354..4370
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4378..4384,4385..4402,4403..4408))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4423..4444
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4503..4698
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4549..4684
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4704..4838
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4741..4760)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4795..4814
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4906..4923)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5077..5665)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5157..5176)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5836..6627,6628..6696))
                     /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     6459..6478
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6697..6801)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg
      541 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      601 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      661 ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc
      721 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
      781 ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct gcgtgaaagc
      841 cttaaagggc tccgggaggg ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg
      901 tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc
      961 gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc
     1081 gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gcccccggag cgccggcggc
     1321 tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag
     1381 ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc
     1441 tctagcgggc gcggggcgaa gcggtgcggc gccggcagga aggaaatggg cggggagggc
     1501 cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg
     1561 ggggacggct gccttcgggg gggacggggc agggcggggt tcggcttctg gcgtgtgacc
     1621 ggcggctcta gagcctctgc taaccatgtt catgccttct tctttttcct acagctcctg
     1681 ggcaacgtgc tggttattgt gctgtctcat cattttggca aagaattccc ataacttcgt
     1741 ataaagtatc ctatacgaag ttatatcaaa ataggaagac caatgcttca ccatcgaccc
     1801 gaattgccaa gcatcaccat cgacccataa cttcgtataa tgtatgctat acgaagttat
     1861 actagctagc tcattacttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca
     1921 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact
     1981 gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct
     2041 gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt
     2101 tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt
     2161 tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct
     2221 cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc
     2281 cgtcgtcctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga
     2341 agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca
     2401 gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcaa atgaacttca
     2461 gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc
     2521 cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc
     2581 ccttgctcac tgctactacc ggtgccgcca ctcctccctc ctcctcctcg tcgacgaagg
     2641 tctcgatctc catgttctcg ccggccacgt tgatggtggt tgtcttccgg atgtcgccgt
     2701 ggatcaggat gtgctcgtgg atcttcactc tcaggtggtg gcccaggaag ccccaagtgt
     2761 tcttgctgat cagatccagg atcaggtggc cgattccgct ggtgtaaggt gtgatcaggc
     2821 ccagtccctc gtgtccggcg atgaacatca cggggaacat cagccaagag cagaagaaga
     2881 cgtaggccat gatcttgcaa atcttccggc agacgccttt gggcagggtg tggtagctct
     2941 cgatatacac cttggcgatc tggaagaagc aagtcacgcc gtagaacagg ccgatcatga
     3001 agaacaggat cttcaggggg ccctttgtaa aggcggctgt gatcccccac acgatgttgc
     3061 cgatgtcggt caccaggatg gtcatggtcc gcttgctgta ctcttcgtgc agtccggtca
     3121 ggttgctcag gtggatcagg atcacggggc aagtcaggag ccacatgctg taccgcagcc
     3181 aaatcacggc tcctccgttg gtctggtaca ctgtggctgg gctgtcgacc tcgtggaaca
     3241 gctcgatgaa gcacttgacg agctcgatca cgcagacgta gacctcctcc cagccggttg
     3301 tggccttgaa gctgtgccaa ccgtagaaga tgagctgcac gatagccacg atcacggtga
     3361 accactggag tcccactgcg atcttgtgct gcagctcggt tccgtggttg atgtggggga
     3421 agcaaccgtg atcggctccg gctgttgtgg cgttagaggt ttctccgtga gcgtctgctg
     3481 ctggtgtcac cactgctgcg gcgctcagca ggcctctaat tgtggcttcg gcgctaggca
     3541 cggctgagat aaaggcgtgg gtcattgtgg cggctgtttc catggtggcg gcgcggccta
     3601 ggtacctagt ataacttcgt ataggatact ttatacgaag ttatcattgg gattcttcct
     3661 attttgatcc aagcatcacc atcgaccctc tagtccagat ctcaccatcg acccataact
     3721 tcgtatagca tacattatac gaagttatgt ccctcgaaga ggttcgcggc cgcactcctc
     3781 aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg gctcacaaat
     3841 accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag ccccttgagc
     3901 atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt tggaattttt
     3961 tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat cagaatgagt
     4021 atttggttta gagtttggca acatatgcca tatgctggct gccatgaaca aaggtggcta
     4081 taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt ccatagaaaa
     4141 gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt ttctttaaca
     4201 tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc ctgactactc
     4261 ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc caagcttggc
     4321 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4381 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4441 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat
     4501 ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     4561 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     4621 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     4681 ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt tacaaataaa
     4741 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     4801 tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat gaatcggcca
     4861 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     4921 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     4981 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5041 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5101 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5161 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5221 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5281 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5341 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5401 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5461 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     5521 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     5581 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     5641 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     5701 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     5761 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     5821 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     5881 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     5941 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6001 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6061 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6121 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6181 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6241 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6301 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6361 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6421 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6481 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     6541 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     6601 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     6661 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     6721 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     6781 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg
//
