LOCUS       sequence-239176-        6926 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Natively produces Halotag, 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 6926)
  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 6926)
  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 Halotag to mScarlet
FEATURES             Location/Qualifiers
     source          1..6926
                     /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             1779..2669
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     polyA_signal    2741..2796
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     protein_bind    3112..3145
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3152..3847
                     /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(3285..3303)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3553..3572
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3863..3919
                     /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    4022..4077
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4429..4445)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4429..4445)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4442..4464)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4453..4469
                     /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(4477..4483,4484..4501,4502..4507))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4522..4543
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4602..4797
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4648..4783
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4803..4937
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4840..4859)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4894..4913
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5005..5022)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5176..5764)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5256..5275)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5935..6726,6727..6795))
                     /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     6558..6577
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6796..6900)
                     /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 cgcgtatgat ggcagaaatc ggtactggct
     1801 ttccattcga cccccattat gtggaagtcc tgggcgagcg catgcactac gtcgatgttg
     1861 gtccgcgcga tggcacccct gtgctgttcc tgcacggtaa cccgacctcc tcctacgtgt
     1921 ggcgcaacat catcccgcat gttgcaccga cccatcgctg cattgctcca gacctgatcg
     1981 gtatgggcaa atccgacaaa ccagacctgg gttatttctt cgacgaccac gtccgcttca
     2041 tggatgcctt catcgaagcc ctgggtctgg aagaggtcgt cctggtcatt cacgactggg
     2101 gctccgctct gggtttccac tgggccaagc gcaatccaga gcgcgtcaaa ggtattgcat
     2161 ttatggagtt catccgccct atcccgacct gggacgaatg gccagaattt gcccgcgaga
     2221 ccttccaggc cttccgcacc accgacgtcg gccgcaagct gatcatcgat cagaacgttt
     2281 ttatcgaggg tacgctgccg atgggtgtcg tccgcccgct gactgaagtc gagatggacc
     2341 attaccgcga gccgttcctg aatcctgttg accgcgagcc actgtggcgc ttcccaaacg
     2401 agctgccaat cgccggtgag ccagcgaaca tcgtcgcgct ggtcgaagaa tacatggact
     2461 ggctgcacca gtcccctgtc ccgaagctgc tgttctgggg caccccaggc gttctgatcc
     2521 caccggccga agccgctcgc ctggccaaaa gcctgcctaa ctgcaaggct gtggacatcg
     2581 gcccgggtct gaatctgctg caagaagaca acccggacct gatcggcagc gagatcgcgc
     2641 gctggctgtc gacgctcgag atttccggct aaagatcttt ttccctctgc caaaaattat
     2701 ggggacatca tgaagcccct tgagcatctg acttctggct aataaaggaa atttattttc
     2761 attgcaatag tgtgttggaa ttttttgtgt ctctcactcg gaaggacata tgggagggca
     2821 aatcatttaa aacatcagaa tgagtatttg gtttagagtt tggcaacata tgcccatatg
     2881 ctggctgcca tgaacaaagg ttggctataa agaggtcatc agtatatgaa acagccccct
     2941 gctgtccatt ccttattcca tagaaaagcc ttgacttgag gttagatttt ttttatattt
     3001 tgttttgtgt tatttttttc tttaacatcc ctaaaatttt ccttacatgt tttactagcc
     3061 agatttttcc tcctctcctg actactccca gtcatagctg tccctcttct cataacttcg
     3121 tataatgtat gctatacgaa gttatggtac catggtgagc aagggcgagg cagtgatcaa
     3181 ggagttcatg cggttcaagg tgcacatgga gggctccatg aacggccacg agttcgagat
     3241 cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc tgaaggtgac
     3301 caagggtggc cccctgccct tctcctggga catcctgtcc cctcagttca tgtacggctc
     3361 cagggccttc atcaagcacc ccgccgacat ccccgactac tataagcagt ccttccccga
     3421 gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgccgtga ccgtgaccca
     3481 ggacacctcc ctggaggacg gcaccctgat ctacaaggtg aagctccgcg gcaccaactt
     3541 ccctcctgac ggccccgtaa tgcagaagaa gacaatgggc tgggaagcgt ccaccgagcg
     3601 gttgtacccc gaggacggcg tgctgaaggg cgacattaag atggccctgc gcctgaagga
     3661 cggcggacgc tacctggcgg acttcaagac cacctacaag gccaagaagc ccgtgcagat
     3721 gcccggcgcc tacaacgtcg accgcaagtt ggacatcacc tcccacaacg aggactacac
     3781 cgtggtggaa cagtacgaac gctccgaggg ccgccactcc accggcggca tggacgagct
     3841 gtacaagttc gaaggcagcg gcgctactaa cttcagcctg ctgaagcagg ctggagacgt
     3901 ggaagagaat cctggaccta tcgataccgg tcaattggcg gccgcgcatg cgctagctaa
     3961 tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc
     4021 taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc
     4081 ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt
     4141 ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat
     4201 cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga
     4261 ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt
     4321 tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct
     4381 gtccctcttc tcttatggag atccctcgac aagcttggcg taatcatggt catagctgtt
     4441 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     4501 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     4561 gcccgctttc cagtcgggaa acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca
     4621 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4681 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     4741 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     4801 ctaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca
     4861 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat
     4921 cttatcatgt ctggatccgc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     4981 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     5041 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     5101 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     5161 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     5221 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     5281 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     5341 tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt
     5401 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     5461 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     5521 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     5581 cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct
     5641 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     5701 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     5761 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     5821 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     5881 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     5941 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     6001 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     6061 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     6121 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     6181 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     6241 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     6301 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     6361 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     6421 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     6481 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     6541 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     6601 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     6661 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     6721 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     6781 atgttgaata ctcatactct tcctttttca atattattga agcatttatc agggttattg
     6841 tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     6901 cacatttccc cgaaaagtgc cacctg
//
