LOCUS       sequence-182712-        8240 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of cytosolic green-to-red photoconvertible 
            Dendra2 and the nuclear blue fluorescent protein Cerulean..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8240)
  AUTHORS   Kalyviotis K, Qin H, Pantazis P
  TITLE     PhOTO zebrafish and primed conversion: advancing the mechanistic 
            view of development and disease
  JOURNAL   (2020) Behavioral and Neural Genetics of Zebrafish, 309–322.
REFERENCE   2  (bases 1 to 8240)
  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: "(2020) 
            Behavioral and Neural Genetics of Zebrafish, 309–322."
COMMENT     Sequence Label: PhOTO-C (pMTB:cyto-Dendra2-2A-H2B-Cerulean)
FEATURES             Location/Qualifiers
     source          1..8240
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      353..941
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     842..861
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1095..1112
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1229..1250
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1265..1270,1271..1288,1289..1295)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1303..1319
                     /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)."
     primer_bind     1308..1330
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1327..1343
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1327..1343
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1362..1382
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1364..1382
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1419..1435
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1419..1435
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     misc_feature    join(1469..1480,1481..1668)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     promoter        4383..4401
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     4383..4400
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             4443..5132
                     /codon_start=1
                     /product="monomeric photoswitchable green-to-red 
                     fluorescent protein from Dendronephthya"
                     /label=Dendra2
                     /note="mammalian codon-optimized"
                     /translation="MNTPGINLIKEDMRVKVHMEGNVNGHAFVIEGEGKGKPYEGTQTA
                     NLTVKEGAPLPFSYDILTTAVHYGNRVFTKYPEDIPDYFKQSFPEGYSWERTMTFEDKG
                     ICTIRSDISLEGDCFFQNVRFKGTNFPPNGPVMQKKTLKWEPSTEKLHVRDGLLVGNIN
                     MALLLEGGGHYLCDFKTTYKAKKVVQLPDAHFVDHRIEILGNDSDYNKVKLYEHAVARY
                     SPLPSQVW"
     CDS             5178..5231
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             5238..5615
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      5628..5637
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(5634..5636,5637..5639,5640..6353)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Rizzo et al., 
                     2004)"
                     /label=mCerulean
                     /note=" mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNAISDNVYITADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(5679..5700)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5940..5959)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6287..6308
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(6360..6378)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     polyA_signal    6385..6519
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6409..6428)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6463..6482
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     misc_feature    join(6611..6748,6749..6760)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(6790..6809)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6791..6809)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6819..6836)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6819..6835)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6828..6850)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6977..7431
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7063..7082)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7273..7294
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        7457..7561
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(7562..7630,7631..8240,1..182)
                     /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(7780..7799)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
       61 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
      121 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
      181 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
      241 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
      301 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
      361 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
      421 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
      481 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
      541 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
      601 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
      661 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
      721 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
      781 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
      841 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
      901 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
      961 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     1021 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     1081 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     1141 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     1201 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     1261 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     1321 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa
     1381 gggaacaaaa gctggagctc caccgcggtg gcggccgctc tagaactagt ggatctgctg
     1441 ggcttgctga aggtaggggg tcaagaacca gaggtgtaaa gtacttgagt aattttactt
     1501 gattactgta cttaagtatt atttttgggg atttttactt tacttgagta caattaaaaa
     1561 tcaatacttt tacttttact taattacatt tttttagaaa aaaaagtact ttttactcct
     1621 tacaatttta tttacagtca aaaagtactt attttttgga gatcacttgg gcccggctcg
     1681 agggcctagt cattacctat actcaacgtt tactctgaga aacttccata ttttagaggt
     1741 aaaatgctca gaattgtaaa aagtcttgtc aaaatacttg gaatttgcca aaaacaaaat
     1801 aaaagtcaca tcacctcatt gtccttggta tcagcaagca cttttaacac aatcagctgt
     1861 ttttgtttat aactcccact gaaggccatg tgcattactc ccaagttaca cagtaaaaag
     1921 tcacatgtag tttatcatga tcactgctgc tcaagcacag ctttatctgt tattcagtgt
     1981 ggcaatatct gaaatgtgtg caaaatatta tttattgtag ctgatcacca acactttcag
     2041 tgtttttttt tttctacttt taatgagaat tatgttctta ggatttatcc ttataaacct
     2101 ctttaaaaag aaatgttatg tctgcataga cacgacatga aattatgaac agaaaattta
     2161 actatttaag atcaagactt gacatataac atttctaatg aacattaaac ctgaggtagg
     2221 cccacttgca aaaatgtaac tgctatcact cactttatac atcattgtgt aacttgtaca
     2281 cacaaaaata gtgaagcaat gtggcatgga atgcaggtcc cattcattca aagttattaa
     2341 agcactttct taaagtgcat tactactgag ctaagaccag ctagttgctc atacaacata
     2401 ctacaagtag tgtcatccta atttggataa aaaaaatgtc cattttatac atggaaaagt
     2461 actcattaga tcccccattg tttgtattac ggtatttcgt gaacacaaga ggtaaatgac
     2521 ctacagagct gctgttgtgt tagattgaaa acacaacaca ggatcatgga ggcatccatc
     2581 attatgacgc attctgccac ttgtaactct gcacagactt tttgaaaagt ttaattgagt
     2641 cttggcgttg tcgcagaaaa atacgaaaaa ttctgtgtta aaatttgcaa ataaaaattc
     2701 cagttttata ggaaatcgcc atggccttgt tctttaacca gttaagcttc cccttctttc
     2761 actctcaagt tgcaagaagc aagtgtagca atgtgcacgc gacagccggg tgtgtgacgc
     2821 tggaccaatc agagcacaga gctccgaaag tttacctttt atggctagag ccgggtatgt
     2881 gccgtcatat aaaagagcgc gcccagcttt tcagcctcac tttgagctcc tccacacgca
     2941 gctagtgcgg aatatcatct gcttgtaacc cattctctta agtcgacaac cccccaaacc
     3001 caaggtgagt tggttttaca gtttttaagc gtttaattat agtttctatc tattaatgtt
     3061 aagtaaaatg tgtaaatgga taaatatgat ttgatgaagt ctttagacag ttcataacgg
     3121 tctctttgta gttatctact aggtttgatt tataagtggt ggttcttttg ataatctatg
     3181 ttatatttaa taaatattgg ctgtatctta acttttttgt agcgttagtg gatgtggcag
     3241 gtgagaatgt cggtaattta acgtgaccag tcgtaggcac gacattgaac attgaatagg
     3301 ccggtgtgaa atgggtgtta agtcttaata taaagttgtg ttctggctaa tctctcttta
     3361 acagccataa atgtgatttt taactacagt tttgagggtg ctggtgagca tgttgcacac
     3421 ttgatggata gccggcatgg gaagttcttt gtgcaggcag tgctgcagca gggtgtgacc
     3481 tactttagct agccggctaa ccagctctca tctgatgtaa cgtaaacccc atcaatctaa
     3541 catcggctca ttcgtgcttt ataagatcga agatcttgtt attagtaacg ggtattggca
     3601 tttagtttta acttggttaa ttttcttttt gttatgtgtt ctatttaaat gtagcctgca
     3661 gccccctctg tctttaatga agctgaagat taacgttaac gttagtgaag tgttgtatcc
     3721 gctatgaaag atttgaattt tagaggtgtg gtccacttta ataacggctg cggagttctc
     3781 tggctgcact tcagccgctg tgactgtcga agcagagccc atcagttttt caggatctcg
     3841 gtgatttttg aggctagact atgaactgaa ccgactgaga gtgaccgtcc agaaatgctt
     3901 tagtgtgaag tctccgctca cgaggagtcc gtggactgac tgcagatctg tgacgcagta
     3961 acacccggcg gcagacaccc gttgaaatgc ggttgtgtaa ttgatcgcag gcgagggtgg
     4021 aagaggatgt gaaacttcat ttgtgtaaaa tttagggagt ggtcctggtg tgatgaatgt
     4081 cgaaatctgt tcctttttac tgagccctac gactctggct gagtgccaca ccgccggcag
     4141 ccgcaaagcg gctcaaacca ttgcctttta tggtaataat gagagaatgc agagggactt
     4201 cctttgtctg gcatatctga ggcgcgcgct gtcactagcg cccaccagcg gtcagactgt
     4261 agaatgcaga gcaaaacagg aagttgactc cagatggtca catgcttgct gaaacgcctc
     4321 tccctggcag cagtgcaatt ctaacagttt ccctttcttt tacagttcgc catcctaggc
     4381 ctatttaggt gacactatag aagagtacaa gctacttgtt ctttttgcag gatcccatcg
     4441 atatgaacac cccgggaatt aacctgatca aggaggacat gcgcgtgaag gtgcacatgg
     4501 agggcaacgt gaacggccac gccttcgtga tcgagggcga gggcaagggc aagccctacg
     4561 agggcaccca gaccgccaac ctgaccgtga aggagggcgc ccccctgccc ttcagctacg
     4621 acatcctgac caccgccgtg cactacggca accgggtgtt caccaagtac cccgaggaca
     4681 tccccgacta cttcaagcag agcttccccg agggctacag ctgggagcgc accatgacct
     4741 tcgaggacaa gggcatctgc accatccgca gcgacatcag cctggagggc gactgcttct
     4801 tccagaacgt gcgcttcaag ggcaccaact tcccccccaa cggccccgtg atgcagaaga
     4861 agaccctgaa gtgggagccc agcaccgaga agctgcacgt gcgcgacggc ctgctggtgg
     4921 gcaacatcaa catggccctg ctgctggagg gcggcggcca ctacctgtgc gacttcaaga
     4981 ccacctacaa ggccaagaag gtggtgcagc tgcccgacgc ccacttcgtg gaccaccgca
     5041 tcgagatcct gggcaacgac agcgactaca acaaggtgaa gctgtacgag cacgccgtgg
     5101 cccgctacag ccccctgccc agccaggtgt ggtccggcga cagcggcgtg tacaagagtt
     5161 taattaaggg atcaggagag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga
     5221 atcccggccc ttacgtaatg ccagagccag cgaagtctgc tcccgccccg aaaaagggct
     5281 ccaagaaggc ggtgactaag gcgcagaaga aaggcggcaa gaagcgcaag cgcagccgca
     5341 aggagagcta ttccatctat gtgtacaagg ttctgaagca ggtccaccct gacaccggca
     5401 tttcgtccaa ggccatgggc atcatgaatt cgtttgtgaa cgacattttc gagcgcatcg
     5461 caggtgaggc ttcccgcctg gcgcattaca acaagcgctc gaccatcacc tccagggaga
     5521 tccagacggc cgtgcgcctg ctgctgcctg gggagttggc caagcacgcc gtgtccgagg
     5581 gtactaaggc catcaccaag tacaccagcg ctaaggatcc acaggtcgcc accatggtga
     5641 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     5701 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     5761 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     5821 ccaccctgac ctggggcgtg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg
     5881 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     5941 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     6001 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     6061 agtacaacgc catcagcgac aacgtctata tcaccgccga caagcagaag aacggcatca
     6121 aggccaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     6181 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     6241 gcacccagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     6301 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag taagacgtcc
     6361 cgctgagcaa taactagcac cggtgatcca gacatgataa gatacattga tgagtttgga
     6421 caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt
     6481 gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat
     6541 tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt gatatcccgg gtttaaacgc
     6601 atgcagatct aatactcaag tacaatttta atggagtact tttttacttt tactcaagta
     6661 agattctagc cagatacttt tacttttaat tgagtaaaat tttccctaag tacttgtact
     6721 ttcacttgag taaaattttt gagtactttt tacacctctg tcaagaacca tatgccggta
     6781 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     6841 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     6901 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     6961 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     7021 acgcgcagcg tgaccccccc acccgccgcg ccctagcgcc cgctcctttc gctttcttcc
     7081 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     7141 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     7201 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     7261 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     7321 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     7381 tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt taggtggcac
     7441 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     7501 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     7561 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     7621 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     7681 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     7741 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     7801 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     7861 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     7921 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     7981 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     8041 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     8101 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     8161 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     8221 ctcggccctt ccggctggct
//
