LOCUS       sequence-238543-        6752 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Natively produces mClover3, in the presence of Cre will be 
            recombined to produce mScarlet..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6752)
  AUTHORS   Taylor RJ, Carrington J, Gerlach LR, Taylor KL, Richters KE, Dent EW
  TITLE     Double UP: A Dual Color, Internally Controlled Platform for in utero
            Knockdown or Overexpression.
  JOURNAL   Front Mol Neurosci. 2020 May 20;13:82. doi: 
            10.3389/fnmol.2020.00082. eCollection 2020.
  PUBMED    32508591
REFERENCE   2  (bases 1 to 6752)
  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: "Front Mol 
            Neurosci."; date: "2020-05-20"; volume: "13:82. doi"; pages: " 
            10.3389/fnmol.2020.00082. eCollection 2020"
COMMENT     Sequence Label: Double UP mClover3 to mScarlet
FEATURES             Location/Qualifiers
     source          1..6752
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1739..1772
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             join(1779..1781,1782..1784,1785..2498)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1824..1845)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2432..2453
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2567..2622
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     protein_bind    2938..2971
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2978..3673
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(3111..3129)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3379..3398
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3689..3745
                     /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"
     polyA_signal    3848..3903
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4255..4271)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4255..4271)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4268..4290)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4279..4295
                     /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(4303..4309,4310..4327,4328..4333))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4348..4369
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4428..4623
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4474..4609
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4629..4763
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4666..4685)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4720..4739
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4831..4848)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5002..5590)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5082..5101)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5761..6552,6553..6621))
                     /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     6384..6403
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6622..6726)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggsgcgc gccrggsggg gsggggsggg gsgrggggsg
      541 gggsggggsg rggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgctgcagat
     1741 aacttcgtat aatgtatgct atacgaagtt atacgcgtat ggtgagcaag ggcgaggagc
     1801 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1861 tcagcgtccg cggcgagggc gagggcgatg ccaccaacgg caagctgacc ctgaagttca
     1921 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ttcggctacg
     1981 gcgtggcctg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     2041 ccatgcccga aggctacgtc caggagcgca ccatctcttt caaggacgac ggtacctaca
     2101 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     2161 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aacttcaaca
     2221 gccactacgt ctatatcacg gccgacaagc agaagaactg catcaaggct aacttcaaga
     2281 tccgccacaa cgttgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc
     2341 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagccat cagtccaagc
     2401 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg
     2461 ccgggattac acatggcatg gacgagctgt acaagtaaag atctttttcc ctctgccaaa
     2521 aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt
     2581 attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag gacatatggg
     2641 agggcaaatc atttaaaaca tcagaatgag tatttggttt agagtttggc aacatatgcc
     2701 catatgctgg ctgccatgaa caaaggttgg ctataaagag gtcatcagta tatgaaacag
     2761 ccccctgctg tccattcctt attccataga aaagccttga cttgaggtta gatttttttt
     2821 atattttgtt ttgtgttatt tttttcttta acatccctaa aattttcctt acatgtttta
     2881 ctagccagat ttttcctcct ctcctgacta ctcccagtca tagctgtccc tcttctcata
     2941 acttcgtata atgtatgcta tacgaagtta tggtaccatg gtgagcaagg gcgaggcagt
     3001 gatcaaggag ttcatgcggt tcaaggtgca catggagggc tccatgaacg gccacgagtt
     3061 cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg ccaagctgaa
     3121 ggtgaccaag ggtggccccc tgcccttctc ctgggacatc ctgtcccctc agttcatgta
     3181 cggctccagg gccttcatca agcaccccgc cgacatcccc gactactata agcagtcctt
     3241 ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg ccgtgaccgt
     3301 gacccaggac acctccctgg aggacggcac cctgatctac aaggtgaagc tccgcggcac
     3361 caacttccct cctgacggcc ccgtaatgca gaagaagaca atgggctggg aagcgtccac
     3421 cgagcggttg taccccgagg acggcgtgct gaagggcgac attaagatgg ccctgcgcct
     3481 gaaggacggc ggacgctacc tggcggactt caagaccacc tacaaggcca agaagcccgt
     3541 gcagatgccc ggcgcctaca acgtcgaccg caagttggac atcacctccc acaacgagga
     3601 ctacaccgtg gtggaacagt acgaacgctc cgagggccgc cactccaccg gcggcatgga
     3661 cgagctgtac aagttcgaag gcagcggcgc tactaacttc agcctgctga agcaggctgg
     3721 agacgtggaa gagaatcctg gacctatcga taccggtcaa ttggcggccg cgcatgcgct
     3781 agctaatttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     3841 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     3901 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     3961 tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga
     4021 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg
     4081 acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta
     4141 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc
     4201 atagctgtcc ctcttctctt atggagatcc ctcgacaagc ttggcgtaat catggtcata
     4261 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     4321 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4381 ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca tctcaattag
     4441 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4501 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4561 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4621 aaaaagctaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4681 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4741 atgtatctta tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg cggggagagg
     4801 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4861 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4921 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4981 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     5041 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     5101 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     5161 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     5221 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     5281 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     5341 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5401 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5461 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5521 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5581 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5641 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5701 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5761 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5821 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5881 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5941 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6001 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6061 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6121 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6181 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6241 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6301 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6361 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6421 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6481 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6541 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6601 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6661 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6721 tccgcgcaca tttccccgaa aagtgccacc tg
//
