LOCUS       sequence-239175-        7307 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 7307)
  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 7307)
  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..7307
                     /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             1770..2480
                     /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      2025..2034
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2549..2604
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     protein_bind    2920..2953
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             2960..3655
                     /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(3093..3111)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3361..3380
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3671..3727
                     /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(3740..3787,3788..3790,3791..4318)
                     /codon_start=1
                     /product="dominant negative mutant of human Rac family 
                     small GTPase 1"
                     /label=Rac1 (T17N)
                     /translation="MQAIKCVVVGDGAVGKNCLLISYTTNAFPGEYIPTVFDNYSANVM
                     VDGKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASFENVRAKWYPEVRHH
                     CPNTPIILVGTKLDLRDDKDTIEKLKEKKLTPITYPQGLAMAKEIGAVKYLECSALTQR
                     GLKTVFDEAIRAVLCPPPVKKRKRKCLLL"
     polyA_signal    4403..4458
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4810..4826)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4810..4826)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4823..4845)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4834..4850
                     /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(4858..4864,4865..4882,4883..4888))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4903..4924
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4983..5178
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5029..5164
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5184..5318
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5221..5240)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5275..5294
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5386..5403)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5557..6145)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5637..5656)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6316..7107,7108..7176))
                     /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     6939..6958
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7177..7281)
                     /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 tggtgagcaa gggcgaggag gataacatgg
     1801 cctctctccc agcgacacat gagttacaca tctttggctc catcaacggt gtggactttg
     1861 acatggtggg tcagggcacc ggcaatccaa atgatggtta tgaggagtta aacctgaagt
     1921 ccaccaaggg tgacctccag ttctccccct ggattctggt ccctcatatc gggtatggct
     1981 tccatcagta cctgccctac cctgacggga tgtcgccttt ccaggccgcc atggtagatg
     2041 gctccggcta ccaagtccat cgcacaatgc agtttgaaga tggtgcctcc cttactgtta
     2101 actaccgcta cacctacgag ggaagccaca tcaaaggaga ggcccaggtg aaggggactg
     2161 gtttccctgc tgacggtcct gtgatgacca actcgctgac cgctgcggac tggtgcaggt
     2221 cgaagaagac ttaccccaac gacaaaacca tcatcagtac ctttaagtgg agttacacca
     2281 ctggaaatgg caagcgctac cggagcactg cgcggaccac ctacaccttt gccaagccaa
     2341 tggcggctaa ctatctgaag aaccagccga tgtacgtgtt ccgtaagacg gagctcaagc
     2401 actccaagac cgagctcaac ttcaaggagt ggcaaaaggc ctttaccgat gtgatgggca
     2461 tggacgagct gtacaagtaa agatcttttt ccctctgcca aaaattatgg ggacatcatg
     2521 aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat tgcaatagtg
     2581 tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa tcatttaaaa
     2641 catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct ggctgccatg
     2701 aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc tgtccattcc
     2761 ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg ttttgtgtta
     2821 tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag atttttcctc
     2881 ctctcctgac tactcccagt catagctgtc cctcttctca taacttcgta taatgtatgc
     2941 tatacgaagt tatggtacca tggtgagcaa gggcgaggca gtgatcaagg agttcatgcg
     3001 gttcaaggtg cacatggagg gctccatgaa cggccacgag ttcgagatcg agggcgaggg
     3061 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc
     3121 cctgcccttc tcctgggaca tcctgtcccc tcagttcatg tacggctcca gggccttcat
     3181 caagcacccc gccgacatcc ccgactacta taagcagtcc ttccccgagg gcttcaagtg
     3241 ggagcgcgtg atgaacttcg aggacggcgg cgccgtgacc gtgacccagg acacctccct
     3301 ggaggacggc accctgatct acaaggtgaa gctccgcggc accaacttcc ctcctgacgg
     3361 ccccgtaatg cagaagaaga caatgggctg ggaagcgtcc accgagcggt tgtaccccga
     3421 ggacggcgtg ctgaagggcg acattaagat ggccctgcgc ctgaaggacg gcggacgcta
     3481 cctggcggac ttcaagacca cctacaaggc caagaagccc gtgcagatgc ccggcgccta
     3541 caacgtcgac cgcaagttgg acatcacctc ccacaacgag gactacaccg tggtggaaca
     3601 gtacgaacgc tccgagggcc gccactccac cggcggcatg gacgagctgt acaagttcga
     3661 aggcagcggc gctactaact tcagcctgct gaagcaggct ggagacgtgg aagagaatcc
     3721 tggacctatc gataccggta tgcaggccat caagtgtgtg gtggtgggag acggagctgt
     3781 aggtaaaaac tgcctactga tcagttacac aaccaatgca tttcctggag aatatatccc
     3841 tactgtcttt gacaattatt ctgccaatgt tatggtagat ggaaaaccgg tgaatctggg
     3901 cttatgggat acagctggac aagaagatta tgacagatta cgccccctat cctatccgca
     3961 aacagatgtg ttcttaattt gcttttccct tgtgagtcct gcatcatttg aaaatgtccg
     4021 tgcaaagtgg tatcctgagg tgcggcacca ctgtcccaac actcccatca tcctagtggg
     4081 aactaaactt gatcttaggg atgataaaga cacgatcgag aaactgaagg agaagaagct
     4141 gactcccatc acctatccgc agggtctagc catggctaag gagattggtg ctgtaaaata
     4201 cctggagtgc tcggcgctca cacagcgagg cctcaagaca gtgtttgacg aagcgatccg
     4261 agcagtcctc tgcccgcctc ccgtgaagaa gaggaagaga aaatgcctgc tgttgtaagc
     4321 ggccgcgcat gcgctagcta atttccctct gccaaaaatt atggggacat catgaagccc
     4381 cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg
     4441 aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt aaaacatcag
     4501 aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc catgaacaaa
     4561 ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca ttccttattc
     4621 catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt gttatttttt
     4681 tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt cctcctctcc
     4741 tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga caagcttggc
     4801 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4861 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4921 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagcggat
     4981 ccgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     5041 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     5101 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     5161 ggcctaggct tttgcaaaaa gctaacttgt ttattgcagc ttataatggt tacaaataaa
     5221 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     5281 tgtccaaact catcaatgta tcttatcatg tctggatccg ctgcattaat gaatcggcca
     5341 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5401 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5461 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5521 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5581 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5641 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5701 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg
     5761 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     5821 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     5881 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     5941 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
     6001 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     6061 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     6121 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     6181 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     6241 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     6301 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     6361 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     6421 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     6481 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     6541 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     6601 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     6661 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     6721 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     6781 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     6841 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     6901 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     6961 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7021 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7081 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7141 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7201 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7261 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctg
//
