LOCUS       sequence-238540-        6753 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Natively produces sfGFP, 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 6753)
  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 6753)
  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 sfGFP to mScarlet
FEATURES             Location/Qualifiers
     source          1..6753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          663..1679
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1603..1625
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1687..1706
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     protein_bind    1740..1773
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             join(1780..1782,1783..1785,1786..2499)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1825..1846)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     2433..2454
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2568..2623
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     protein_bind    2939..2972
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2979..3674
                     /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(3112..3130)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3380..3399
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3690..3746
                     /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    3849..3904
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4256..4272)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4256..4272)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4269..4291)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4280..4296
                     /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(4304..4310,4311..4328,4329..4334))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4349..4370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4429..4624
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4475..4610
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4630..4764
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4667..4686)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4721..4740
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4832..4849)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5003..5591)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5083..5102)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5762..6553,6554..6622))
                     /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     6385..6404
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6623..6727)
                     /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 gggggnsgcg cgccrggsgg ggsggggsgg ggsgrggggs
      541 ggggsggggs grggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
      601 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      661 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
      841 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
      901 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     1021 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     1081 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     1141 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     1201 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     1261 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
     1321 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     1381 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     1441 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     1501 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     1561 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     1621 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     1681 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgctgcaga
     1741 taacttcgta taatgtatgc tatacgaagt tatacgcgta tggtgagcaa gggcgaggag
     1801 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1861 ttcagcgtgc gcggcgaggg cgagggcgat gccaccaacg gcaagctgac cctgaagttc
     1921 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1981 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc gccacgactt cttcaagtcc
     2041 gccatgcccg aaggctacgt ccaggagcgc accatcagct tcaaggacga cggcacctac
     2101 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2161 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caacttcaac
     2221 agccacaacg tctatatcac cgccgacaag cagaagaacg gcatcaaggc caacttcaag
     2281 atccgccaca acgtggagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     2341 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgtg
     2401 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     2461 gccgggatca ctcacggcat ggacgagctg tacaagtaaa gatctttttc cctctgccaa
     2521 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt
     2581 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg
     2641 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc
     2701 ccatatgctg gctgccatga acaaaggttg gctataaaga ggtcatcagt atatgaaaca
     2761 gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt
     2821 tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt
     2881 actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctcat
     2941 aacttcgtat aatgtatgct atacgaagtt atggtaccat ggtgagcaag ggcgaggcag
     3001 tgatcaagga gttcatgcgg ttcaaggtgc acatggaggg ctccatgaac ggccacgagt
     3061 tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga
     3121 aggtgaccaa gggtggcccc ctgcccttct cctgggacat cctgtcccct cagttcatgt
     3181 acggctccag ggccttcatc aagcaccccg ccgacatccc cgactactat aagcagtcct
     3241 tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gccgtgaccg
     3301 tgacccagga cacctccctg gaggacggca ccctgatcta caaggtgaag ctccgcggca
     3361 ccaacttccc tcctgacggc cccgtaatgc agaagaagac aatgggctgg gaagcgtcca
     3421 ccgagcggtt gtaccccgag gacggcgtgc tgaagggcga cattaagatg gccctgcgcc
     3481 tgaaggacgg cggacgctac ctggcggact tcaagaccac ctacaaggcc aagaagcccg
     3541 tgcagatgcc cggcgcctac aacgtcgacc gcaagttgga catcacctcc cacaacgagg
     3601 actacaccgt ggtggaacag tacgaacgct ccgagggccg ccactccacc ggcggcatgg
     3661 acgagctgta caagttcgaa ggcagcggcg ctactaactt cagcctgctg aagcaggctg
     3721 gagacgtgga agagaatcct ggacctatcg ataccggtca attggcggcc gcgcatgcgc
     3781 tagctaattt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
     3841 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
     3901 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
     3961 ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag
     4021 aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt
     4081 gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct
     4141 aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt
     4201 catagctgtc cctcttctct tatggagatc cctcgacaag cttggcgtaa tcatggtcat
     4261 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4321 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4381 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta
     4441 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4501 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4561 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4621 caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4681 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4741 aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag
     4801 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4861 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4921 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4981 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5041 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5101 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5161 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5221 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5281 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5341 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5401 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5461 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5521 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5581 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5641 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5701 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5761 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5821 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5881 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5941 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6001 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6061 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6121 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6181 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6241 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6301 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6361 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6421 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6481 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6541 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6601 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6661 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6721 ttccgcgcac atttccccga aaagtgccac ctg
//
