LOCUS       sequence-244989-        6929 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 6929)
  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 6929)
  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..6929
                     /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             1782..2672
                     /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    2744..2799
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     protein_bind    3115..3148
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             3155..3850
                     /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(3288..3306)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3556..3575
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3866..3922
                     /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    4025..4080
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4432..4448)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4432..4448)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4445..4467)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4456..4472
                     /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(4480..4486,4487..4504,4505..4510))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4525..4546
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4605..4800
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4651..4786
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4806..4940
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4843..4862)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4897..4916
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5008..5025)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5179..5767)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5259..5278)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5938..6729,6730..6798))
                     /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     6561..6580
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6799..6903)
                     /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 aggcggagag 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 gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg rgggctcggg ggaggggcgc 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 atacgcgtat gatggcagaa atcggtactg
     1801 gctttccatt cgacccccat tatgtggaag tcctgggcga gcgcatgcac tacgtcgatg
     1861 ttggtccgcg cgatggcacc cctgtgctgt tcctgcacgg taacccgacc tcctcctacg
     1921 tgtggcgcaa catcatcccg catgttgcac cgacccatcg ctgcattgct ccagacctga
     1981 tcggtatggg caaatccgac aaaccagacc tgggttattt cttcgacgac cacgtccgct
     2041 tcatggatgc cttcatcgaa gccctgggtc tggaagaggt cgtcctggtc attcacgact
     2101 ggggctccgc tctgggtttc cactgggcca agcgcaatcc agagcgcgtc aaaggtattg
     2161 catttatgga gttcatccgc cctatcccga cctgggacga atggccagaa tttgcccgcg
     2221 agaccttcca ggccttccgc accaccgacg tcggccgcaa gctgatcatc gatcagaacg
     2281 tttttatcga gggtacgctg ccgatgggtg tcgtccgccc gctgactgaa gtcgagatgg
     2341 accattaccg cgagccgttc ctgaatcctg ttgaccgcga gccactgtgg cgcttcccaa
     2401 acgagctgcc aatcgccggt gagccagcga acatcgtcgc gctggtcgaa gaatacatgg
     2461 actggctgca ccagtcccct gtcccgaagc tgctgttctg gggcacccca ggcgttctga
     2521 tcccaccggc cgaagccgct cgcctggcca aaagcctgcc taactgcaag gctgtggaca
     2581 tcggcccggg tctgaatctg ctgcaagaag acaacccgga cctgatcggc agcgagatcg
     2641 cgcgctggct gtcgacgctc gagatttccg gctaaagatc tttttccctc tgccaaaaat
     2701 tatggggaca tcatgaagcc ccttgagcat ctgacttctg gctaataaag gaaatttatt
     2761 ttcattgcaa tagtgtgttg gaattttttg tgtctctcac tcggaaggac atatgggagg
     2821 gcaaatcatt taaaacatca gaatgagtat ttggtttaga gtttggcaac atatgcccat
     2881 atgctggctg ccatgaacaa aggttggcta taaagaggtc atcagtatat gaaacagccc
     2941 cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata
     3001 ttttgttttg tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta
     3061 gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcataact
     3121 tcgtataatg tatgctatac gaagttatgg taccatggtg agcaagggcg aggcagtgat
     3181 caaggagttc atgcggttca aggtgcacat ggagggctcc atgaacggcc acgagttcga
     3241 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     3301 gaccaagggt ggccccctgc ccttctcctg ggacatcctg tcccctcagt tcatgtacgg
     3361 ctccagggcc ttcatcaagc accccgccga catccccgac tactataagc agtccttccc
     3421 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggcgccg tgaccgtgac
     3481 ccaggacacc tccctggagg acggcaccct gatctacaag gtgaagctcc gcggcaccaa
     3541 cttccctcct gacggccccg taatgcagaa gaagacaatg ggctgggaag cgtccaccga
     3601 gcggttgtac cccgaggacg gcgtgctgaa gggcgacatt aagatggccc tgcgcctgaa
     3661 ggacggcgga cgctacctgg cggacttcaa gaccacctac aaggccaaga agcccgtgca
     3721 gatgcccggc gcctacaacg tcgaccgcaa gttggacatc acctcccaca acgaggacta
     3781 caccgtggtg gaacagtacg aacgctccga gggccgccac tccaccggcg gcatggacga
     3841 gctgtacaag ttcgaaggca gcggcgctac taacttcagc ctgctgaagc aggctggaga
     3901 cgtggaagag aatcctggac ctatcgatac cggtcaattg gcggccgcgc atgcgctagc
     3961 taatttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct
     4021 ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca
     4081 ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag
     4141 agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct ataaagaggt
     4201 catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact
     4261 tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa
     4321 ttttccttac atgttttact agccagattt ttcctcctct cctgactact cccagtcata
     4381 gctgtccctc ttctcttatg gagatccctc gacaagcttg gcgtaatcat ggtcatagct
     4441 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4501 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4561 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca
     4621 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4681 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4741 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4801 aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4861 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4921 tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4981 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5041 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5101 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5161 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5221 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5281 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5341 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5401 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5461 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5521 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5581 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5641 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5701 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5761 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5821 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5881 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5941 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6001 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6061 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6121 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6181 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6241 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6301 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6361 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6421 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6481 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6541 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6601 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6661 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6721 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6781 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6841 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6901 gcgcacattt ccccgaaaag tgccacctg
//
