LOCUS       sequence-244754-        7310 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Natively produces mNeonGreen, in the presence of Cre will be 
            recombined to produce mScarlet and an untagged Rac1-N17 (Dominant 
            Negative)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7310)
  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 7310)
  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 mNeon to mScarlet Rac1-N17
FEATURES             Location/Qualifiers
     source          1..7310
                     /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             1773..2483
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     regulatory      2028..2037
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2552..2607
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     protein_bind    2923..2956
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2963..3658
                     /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(3096..3114)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3364..3383
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3674..3730
                     /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"
     CDS             join(3743..3790,3791..3793,3794..4321)
                     /codon_start=1
                     /product="dominant negative mutant of human Rac family 
                     small GTPase 1"
                     /label=Rac1 (T17N)
                     /translation="MQAIKCVVVGDGAVGKNCLLISYTTNAFPGEYIPTVFDNYSANVM
                     VDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     CDS             3743..4321
                     /codon_start=1
                     /product="human Rac family small GTPase 1"
                     /label=Rac1
                     /translation="MQAIKCVVVGDGAVGKNCLLISYTTNAFPGEYIPTVFDNYSANVM
                     VDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     polyA_signal    4406..4461
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4813..4829)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4813..4829)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4826..4848)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4837..4853
                     /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(4861..4867,4868..4885,4886..4891))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4906..4927
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4986..5181
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5032..5167
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5187..5321
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5224..5243)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5278..5297
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5389..5406)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5560..6148)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5640..5659)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6319..7110,7111..7179))
                     /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     6942..6961
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7180..7284)
                     /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 gkgggggtgc cgggcggggc
     1261 ggggccgcck cgggccgggg rgggctcggg ggrggggcgc 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 atatggtgag caagggcgag gaggataaca
     1801 tggcctctct cccagcgaca catgagttac acatctttgg ctccatcaac ggtgtggact
     1861 ttgacatggt gggtcagggc accggcaatc caaatgatgg ttatgaggag ttaaacctga
     1921 agtccaccaa gggtgacctc cagttctccc cctggattct ggtccctcat atcgggtatg
     1981 gcttccatca gtacctgccc taccctgacg ggatgtcgcc tttccaggcc gccatggtag
     2041 atggctccgg ctaccaagtc catcgcacaa tgcagtttga agatggtgcc tcccttactg
     2101 ttaactaccg ctacacctac gagggaagcc acatcaaagg agaggcccag gtgaagggga
     2161 ctggtttccc tgctgacggt cctgtgatga ccaactcgct gaccgctgcg gactggtgca
     2221 ggtcgaagaa gacttacccc aacgacaaaa ccatcatcag tacctttaag tggagttaca
     2281 ccactggaaa tggcaagcgc taccggagca ctgcgcggac cacctacacc tttgccaagc
     2341 caatggcggc taactatctg aagaaccagc cgatgtacgt gttccgtaag acggagctca
     2401 agcactccaa gaccgagctc aacttcaagg agtggcaaaa ggcctttacc gatgtgatgg
     2461 gcatggacga gctgtacaag taaagatctt tttccctctg ccaaaaatta tggggacatc
     2521 atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata
     2581 gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta
     2641 aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat gctggctgcc
     2701 atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc tgctgtccat
     2761 tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg
     2821 ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc
     2881 ctcctctcct gactactccc agtcatagct gtccctcttc tcataacttc gtataatgta
     2941 tgctatacga agttatggta ccatggtgag caagggcgag gcagtgatca aggagttcat
     3001 gcggttcaag gtgcacatgg agggctccat gaacggccac gagttcgaga tcgagggcga
     3061 gggcgagggc cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg
     3121 ccccctgccc ttctcctggg acatcctgtc ccctcagttc atgtacggct ccagggcctt
     3181 catcaagcac cccgccgaca tccccgacta ctataagcag tccttccccg agggcttcaa
     3241 gtgggagcgc gtgatgaact tcgaggacgg cggcgccgtg accgtgaccc aggacacctc
     3301 cctggaggac ggcaccctga tctacaaggt gaagctccgc ggcaccaact tccctcctga
     3361 cggccccgta atgcagaaga agacaatggg ctgggaagcg tccaccgagc ggttgtaccc
     3421 cgaggacggc gtgctgaagg gcgacattaa gatggccctg cgcctgaagg acggcggacg
     3481 ctacctggcg gacttcaaga ccacctacaa ggccaagaag cccgtgcaga tgcccggcgc
     3541 ctacaacgtc gaccgcaagt tggacatcac ctcccacaac gaggactaca ccgtggtgga
     3601 acagtacgaa cgctccgagg gccgccactc caccggcggc atggacgagc tgtacaagtt
     3661 cgaaggcagc ggcgctacta acttcagcct gctgaagcag gctggagacg tggaagagaa
     3721 tcctggacct atcgataccg gtatgcaggc catcaagtgt gtggtggtgg gagacggagc
     3781 tgtaggtaaa aattgcctac tgatcagtta cacaaccaat gcatttcctg gagaatatat
     3841 ccctactgtc tttgacaatt attctgccaa tgttatggta gatggaaaac cggtgaatct
     3901 gggcttatgg gatacagctg gacaagaaga ttatgacaga ttacgccccc tatcctatcc
     3961 gcaaacagat gtgttcttaa tttgcttttc ccttgtgagt cctgcatcat ttgaaaatgt
     4021 ccgtgcaaag tggtatcctg aggtgcggca ccactgtccc aacactccca tcatcctagt
     4081 gggaactaaa cttgatctta gggatgataa agacacgatc gagaaactga aggagaagaa
     4141 gctgactccc atcacctatc cgcagggtct agccatggct aaggagattg gtgccgtaaa
     4201 atacctggag tgctcggcgc tcacacagcg aggcctcaag acagtgtttg acgaagcgat
     4261 ccgagcagtc ctctgcccgc ctcccgtgaa gaagaggaag agaaaatgcc tgctgttgta
     4321 agcggccgcg catgcgctag ctaatttccc tctgccaaaa attatgggga catcatgaag
     4381 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     4441 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     4501 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     4561 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     4621 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     4681 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     4741 tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct cgacaagctt
     4801 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
     4861 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     4921 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagcg
     4981 gatccgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     5041 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     5101 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     5161 ggaggcctag gcttttgcaa aaagctaact tgtttattgc agcttataat ggttacaaat
     5221 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     5281 gtttgtccaa actcatcaat gtatcttatc atgtctggat ccgctgcatt aatgaatcgg
     5341 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga
     5401 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat
     5461 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca
     5521 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc
     5581 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata
     5641 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc
     5701 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc
     5761 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga
     5821 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
     5881 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag
     5941 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
     6001 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
     6061 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
     6121 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
     6181 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
     6241 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
     6301 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
     6361 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
     6421 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
     6481 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
     6541 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
     6601 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
     6661 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
     6721 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
     6781 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
     6841 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
     6901 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     6961 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     7021 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     7081 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     7141 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     7201 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     7261 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
//
