LOCUS       sequence-353596-        7610 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  dual plasmid labelling cells of different neuronal population with 
            different fluorophores.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7610)
  AUTHORS   Alfadil E, Bradke F, Dupraz S
  TITLE     In Situ Visualization of Axon Growth and Growth Cone Dynamics in 
            Acute Ex Vivo Embryonic Brain Slice Cultures.
  JOURNAL   J Vis Exp. 2021 Oct 14;(176). doi: 10.3791/63068.
  PUBMED    34723948
REFERENCE   2  (bases 1 to 7610)
  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: "J Vis 
            Exp."; date: "2021-10-14"; volume: "(176). doi"; pages: " 
            10.3791/63068"
COMMENT     Sequence Label: pCAG-lox-rox-STOP-rox-tRFP-pA-lox-ZsGreen-pA
FEATURES             Location/Qualifiers
     source          1..7610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        6..385
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        387..664
                     /label=chicken beta-actin promoter
     intron          665..1681
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1605..1627
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1694..1713
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1694..1712
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1741..1774
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1816..1847
                     /label=rox
                     /bound_moiety="Dre recombinase"
     polyA_signal    2014..2148
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2268..2402
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    2522..2656
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    2685..2716
                     /label=rox
                     /bound_moiety="Dre recombinase"
     CDS             2739..3434
                     /codon_start=1
                     /product="red fluorescent protein from Entacmaea 
                     quadricolor"
                     /label=TurboRFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMKIKV
                     VEGGPLPFAFDILATSFMYGSKAFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVLTA
                     TQDTSFQNGCIIYNVKINGVNFPSNGPVMQKKTRGWEANTEMLYPADGGLRGHSQMALK
                     LVGGGYLHCSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKEADKETYVEQHEMAVAKYC
                     DLPSKLGHR"
     polyA_signal    3456..3680
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     protein_bind    3714..3747
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      3808..3817
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3814..4509
                     /codon_start=1
                     /product="Zoanthus green fluorescent protein"
                     /label=ZsGreen1
                     /note="mammalian codon-optimized"
                     /translation="MAQSKHGLTKEMTMKYRMEGCVDGHKFVITGEGIGYPFKGKQAIN
                     LCVVEGGPLPFAEDILSAAFMYGNRVFTEYPQDIVDYFKNSCPAGYTWDRSFLFEDGAV
                     CICNADITVSVEENCMYHESKFYGVNFPADGPVMKKMTDNWEPSCEKIIPVPKQGILKG
                     DVSMYLLLKDGGRLRCQFDTVYKAKSVPRKMPDWHFIQHKLTREDRSDAKNQKWHLTEH
                     AIASGSALP"
     polyA_signal    4535..4759
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4831..4848)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4831..4847)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4840..4862)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(4988..5443)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5125..5146)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5337..5356
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5470..5574
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5575..5643,5644..6435)
                     /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(5793..5812)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6606..7194
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7095..7114
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7348..7365
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7482..7503
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7518..7523,7524..7541,7542..7548)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7556..7572
                     /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     7561..7583
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7580..7596
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7580..7596
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gattcgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
       61 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
      121 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
      181 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      241 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      301 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      361 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc
      421 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
      481 agcgatgggg gcgggggggg ggggggggcg cgcgccaggs ggggsggggs ggggsgrggg
      541 gsggggsggg gcgaggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      781 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      841 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
      901 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
      961 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
     1021 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
     1081 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     1141 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     1201 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggtggggg tgccgggcgg
     1261 ggcggggccg cctcgggccg gggagggctc gggggagggg cgcggcggcc cccggagcgc
     1321 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     1381 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     1441 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     1501 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     1561 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1621 tgtgaccggc ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca
     1681 gatccttaat taataatacg actcactata gggggtcgac ccgccaccaa tcatttaaat
     1741 ataacttcgt ataatgtatg ctatacgaag ttattcgcga tgaataaatg aaagcttggg
     1801 ggtcgacccg ccacctaact ttaaataatt ggcattattt aaagttatcg cgatgaataa
     1861 atgaaagctt gcagatctgc gactctagag gatctgcgac tctagaggat cataatcagc
     1921 cataccacat ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac
     1981 ctgaaacata aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt
     2041 tacaaataaa gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct
     2101 agttgtggtt tgtccaaact catcaatgta tcttatcatg tctggatctg cgactctaga
     2161 ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     2221 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     2281 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     2341 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     2401 tctgcgactc tagaggatca taatcagcca taccacattt gtagaggttt tacttgcttt
     2461 aaaaaacctc ccacacctcc ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt
     2521 taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     2581 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     2641 ttatcatgtc tggatcccca tcaagctgat ccggaaccct taattaactt taaataattg
     2701 gcattattta aagttaggca gccgccctgc aggccaccat gagcgagctg atcaaggaga
     2761 acatgcacat gaagctgtac atggagggca ccgtgaacaa ccaccacttc aagtgcacat
     2821 ccgagggaga aggcaagccc tacgagggca cccagaccat gaagatcaag gtggtcgagg
     2881 gcggccctct ccccttcgcc ttcgacatcc tggctaccag cttcatgtac ggcagcaaag
     2941 ccttcatcaa ccacacccag ggcatccccg acttctttaa gcagtccttc cctgagggct
     3001 tcacatggga gagaatcacc acatacgaag acgggggcgt gctgaccgct acccaggaca
     3061 ccagcttcca gaacggctgc atcatctaca acgtcaagat caacggggtg aacttcccat
     3121 ccaacggccc tgtgatgcag aagaaaacac gcggctggga ggccaacacc gagatgctgt
     3181 accccgctga cggcggcctg agaggccaca gccagatggc cctgaagctc gtgggcgggg
     3241 gctacctgca ctgctccttc aagaccacat acagatccaa gaaacccgct aagaacctca
     3301 agatgcccgg cttccacttc gtggaccaca gactggaaag aatcaaggag gccgacaaag
     3361 agacctacgt cgagcagcac gagatggctg tggccaagta ctgcgacctc cctagcaaac
     3421 tggggcacag atgagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     3481 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     3541 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     3601 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     3661 ggatgcggtg ggctctatgg ggatccccat caagctgatc cggaaccctt aatataactt
     3721 cgtataatgt atgctatacg aagttattag gtccctcgac ctgcagccca agctagatcg
     3781 aattcggccg gccgatatcg gcgcgccgcc accatggccc agtccaagca cggcctgacc
     3841 aaggagatga ccatgaagta ccgcatggag ggctgcgtgg acggccacaa gttcgtgatc
     3901 accggcgagg gcatcggcta ccccttcaag ggcaagcagg ccatcaacct gtgcgtggtg
     3961 gagggcggcc ccttgccctt cgccgaggac atcttgtccg ccgccttcat gtacggcaac
     4021 cgcgtgttca ccgagtaccc ccaggacatc gtcgactact tcaagaactc ctgccccgcc
     4081 ggctacacct gggaccgctc cttcctgttc gaggacggcg ccgtgtgcat ctgcaacgcc
     4141 gacatcaccg tgagcgtgga ggagaactgc atgtaccacg agtccaagtt ctacggcgtg
     4201 aacttccccg ccgacggccc cgtgatgaag aagatgaccg acaactggga gccctcctgc
     4261 gagaagatca tccccgtgcc caagcagggc atcttgaagg gcgacgtgag catgtacctg
     4321 ctgctgaagg acggtggccg cttgcgctgc cagttcgaca ccgtgtacaa ggccaagtcc
     4381 gtgccccgca agatgcccga ctggcacttc atccagcaca agctgacccg cgaggaccgc
     4441 agcgacgcca agaaccagaa gtggcacctg accgagcacg ccatcgcctc cggctccgcc
     4501 ttgccctgag tcagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct
     4561 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt
     4621 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
     4681 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg
     4741 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc gagatccact
     4801 agttctagcc tcgaggctag agcggccgcc actggccgtc gttttacaac gtcgtgactg
     4861 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
     4921 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
     4981 cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc
     5041 agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag
     5101 accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg
     5161 gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca
     5221 tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa
     5281 gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg
     5341 aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta
     5401 accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg
     5461 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     5521 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     5581 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     5641 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     5701 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     5761 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
     5821 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     5881 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     5941 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     6001 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     6061 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     6121 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     6181 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     6241 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     6301 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     6361 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     6421 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     6481 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     6541 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     6601 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     6661 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     6721 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     6781 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     6841 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     6901 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     6961 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     7021 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     7081 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     7141 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     7201 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     7261 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     7321 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     7381 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     7441 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
     7501 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     7561 agcggataac aatttcacac aggaaacagc tatgaccatg aggcgcgccg
//
