LOCUS       sequence-228116-        6749 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 6749)
  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 6749)
  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..6749
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1675
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1599..1621
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1683..1702
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1736..1769
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             join(1776..1778,1779..1781,1782..2495)
                     /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(1821..1842)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2429..2450
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2564..2619
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     protein_bind    2935..2968
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2975..3670
                     /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(3108..3126)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3376..3395
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3686..3742
                     /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    3845..3900
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4252..4268)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4252..4268)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4265..4287)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4276..4292
                     /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(4300..4306,4307..4324,4325..4330))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4345..4366
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4425..4620
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4471..4606
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4626..4760
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4663..4682)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4717..4736
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4828..4845)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4999..5587)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5079..5098)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5758..6549,6550..6618))
                     /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     6381..6400
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6619..6723)
                     /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 tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtag cggggcgtgg
     1201 cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg
     1261 gccgcctcgg gccggggagg gctcggggga ggggcgcggc ggcccccgga gcgccggcgg
     1321 ctgtcgaggc gcggcgagcc gcagccattg ccttttatgg taatcgtgcg agagggcgca
     1381 gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc
     1441 ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaggaaatgg gcggggaggg
     1501 ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg
     1561 gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac
     1621 cggcggctct agagcctctg ctaaccatgt tcatgccttc ttctttttcc tacagctcct
     1681 gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattgc tgcagataac
     1741 ttcgtataat gtatgctata cgaagttata cgcgtatggt gagcaagggc gaggagctgt
     1801 tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca
     1861 gcgtccgcgg cgagggcgag ggcgatgcca ccaacggcaa gctgaccctg aagttcatct
     1921 gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccttc ggctacggcg
     1981 tggcctgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca
     2041 tgcccgaagg ctacgtccag gagcgcacca tctctttcaa ggacgacggt acctacaaga
     2101 cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca
     2161 tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac ttcaacagcc
     2221 actacgtcta tatcacggcc gacaagcaga agaactgcat caaggctaac ttcaagatcc
     2281 gccacaacgt tgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca
     2341 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagccatcag tccaagctga
     2401 gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg
     2461 ggattacaca tggcatggac gagctgtaca agtaaagatc tttttccctc tgccaaaaat
     2521 tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt
     2581 ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg
     2641 gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat
     2701 atgctggctg ccatgaacaa aggttggcta taaagaggtc atcagtatat gaaacagccc
     2761 cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata
     2821 ttttgttttg tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta
     2881 gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcataact
     2941 tcgtataatg tatgctatac gaagttatgg taccatggtg agcaagggcg aggcagtgat
     3001 caaggagttc atgcggttca aggtgcacat ggagggctcc atgaacggcc acgagttcga
     3061 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     3121 gaccaagggt ggccccctgc ccttctcctg ggacatcctg tcccctcagt tcatgtacgg
     3181 ctccagggcc ttcatcaagc accccgccga catccccgac tactataagc agtccttccc
     3241 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgccg tgaccgtgac
     3301 ccaggacacc tccctggagg acggcaccct gatctacaag gtgaagctcc gcggcaccaa
     3361 cttccctcct gacggccccg taatgcagaa gaagacaatg ggctgggaag cgtccaccga
     3421 gcggttgtac cccgaggacg gcgtgctgaa gggcgacatt aagatggccc tgcgcctgaa
     3481 ggacggcgga cgctacctgg cggacttcaa gaccacctac aaggccaaga agcccgtgca
     3541 gatgcccggc gcctacaacg tcgaccgcaa gttggacatc acctcccaca acgaggacta
     3601 caccgtggtg gaacagtacg aacgctccga gggccgccac tccaccggcg gcatggacga
     3661 gctgtacaag ttcgaaggca gcggcgctac taacttcagc ctgctgaagc aggctggaga
     3721 cgtggaagag aatcctggac ctatcgatac cggtcaattg gcggccgcgc atgcgctagc
     3781 taatttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct
     3841 ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca
     3901 ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag
     3961 agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct ataaagaggt
     4021 catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact
     4081 tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa
     4141 ttttccttac atgttttact agccagattt ttcctcctct cctgactact cccagtcata
     4201 gctgtccctc ttctcttatg gagatccctc gacaagcttg gcgtaatcat ggtcatagct
     4261 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4321 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4381 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca
     4441 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4501 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4561 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4621 aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4681 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4741 tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4801 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4861 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4921 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4981 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5041 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5101 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5161 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc
     5221 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5281 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5341 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5401 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     5461 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5521 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5581 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5641 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5701 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5761 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5821 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5881 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5941 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6001 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6061 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6121 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6181 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6241 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6301 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6361 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6421 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6481 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6541 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6601 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6661 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6721 gcgcacattt ccccgaaaag tgccacctg
//
