LOCUS       sequence-306232-        7246 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral (MSCV) expression of E2-Crimson fluorescent protein .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7246)
  AUTHORS   Mann SE, Zhou Z, Landry LG, Anderson AM, Alkanani AK, Fischer J, 
            Peakman M, Mallone R, Campbell K, Michels AW, Nakayama M
  TITLE     Multiplex T Cell Stimulation Assay Utilizing a T Cell Activation 
            Reporter-Based Detection System.
  JOURNAL   Front Immunol. 2020 Apr 9;11:633. doi: 10.3389/fimmu.2020.00633. 
            eCollection 2020.
  PUBMED    32328071
REFERENCE   2  (bases 1 to 7246)
  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 
            Immunol."; date: "2020-04-9"; volume: "11:633. doi"; pages: " 
            10.3389/fimmu.2020.00633. eCollection 2020"
COMMENT     Sequence Label: pMIII-Neo-E2Crimson
FEATURES             Location/Qualifiers
     source          1..7246
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    189..530
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             597..1013
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     943..965
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     981..997
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1028..1037
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1034..1711
                     /codon_start=1
                     /product="far-red noncytotoxic tetrameric variant of DsRed 
                     fluorescent protein (Strack et al., 2009)"
                     /label=E2-Crimson
                     /note="mammalian codon-optimized"
                     /translation="MDSTENVIKPFMRFKVHMEGSVNGHEFEIEGVGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFFYGSKAYIKHPADIPDYLKQSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGTLIYHVKFIGVNFPSDGPVMQKKTLGWEPSTERNYPRDGVLKGENHM
                     ALKLKGGGHYLCEFKSIYMAKKPVKLPGYHYVDYKLDITSHNEDYTVVEQYERAEARHH
                     LFQ"
     primer_bind     1432..1451
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    1743..2292
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1887..1904)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2114..2133
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2302..3105
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2365..2384)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2975..2994
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             3249..3763
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3925..3941
                     /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(3949..3955,3956..3973,3974..3979))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3994..4015
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4132..4149)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4303..4891)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4383..4402)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5062..5853,5854..5922))
                     /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     5685..5704
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5923..6027)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6095..6113
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6151..6173)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6273..6292
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6486..6508
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6636..6655
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(6855..7246,1..125)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgcgtcgccc gggtacccgt attcccaata aagcctcttg ctgtttgcat ccgaatcgtg
       61 gactcgctga tccttgggag ggtctcctca gattgattga ctgcccacct cgggggtctt
      121 tcatttggag gttccaccga gatttggaga cccctgccca gggaccaccg acccccccgc
      181 cgggaggtaa gctggccagc ggtcgtttcg tgtctgtctc tgtctttgtg cgtgtttgtg
      241 ccggcatcta atgtttgcgc ctgcgtctgt actagttagc taactagctc tgtatctggc
      301 ggacccgtgg tggaactgac gagttctgaa cacccggccg caaccctggg agacgtccca
      361 gggactttgg gggccgtttt tgtggcccga cctgaggaag ggagtcgatg tggaatccga
      421 ccccgtcagg atatgtggtt ctggtaggag acgagaacct aaaacagttc ccgcctccgt
      481 ctgaattttt gctttcggtt tggaaccgaa gccgcgcgtc ttgtctgctg cagcgctgca
      541 gcatcgttct gtgttgtctc tgtctgactg tgtttctgta tttgtctgaa aattagggcc
      601 agactgttac cactccctta agtttgacct taggtcactg gaaagatgtc gagcggatcg
      661 ctcacaacca gtcggtagat gtcaagaaga gacgttgggt taccttctgc tctgcagaat
      721 ggccaacctt taacgtcgga tggccgcgag acggcacctt taaccgagac ctcatcaccc
      781 aggttaagat caaggtcttt tcacctggcc cgcatggaca cccagaccag gtcccctaca
      841 tcgtgacctg ggaagccttg gcttttgacc cccctccctg ggtcaagccc tttgtacacc
      901 ctaagcctcc gcctcctctt cctccatccg ccccgtctct cccccttgaa cctcctcgtt
      961 cgaccccgcc tcgatcctcc ctttatccag ccctcactcc ttctctaggc gccggaattc
     1021 agatctcgcc accatggata gcactgagaa cgtcatcaag cccttcatgc gcttcaaggt
     1081 gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgtgg gcgagggcaa
     1141 gccctacgag ggcacccaga ccgccaagct gcaagtgacc aagggcggcc ccctgccctt
     1201 cgcctgggac atcctgtccc cccagttctt ctacggctcc aaggcgtaca tcaagcaccc
     1261 cgccgacatc cccgactacc tcaagcagtc cttccccgag ggcttcaagt gggagcgcgt
     1321 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     1381 caccctcatc taccacgtga agttcatcgg cgtgaacttc ccctccgacg gccccgtaat
     1441 gcagaagaag actctgggct gggagccctc cactgagcgc aactaccccc gcgacggcgt
     1501 gctgaagggc gagaaccaca tggcgctgaa gctgaagggc ggcggccact acctgtgtga
     1561 gttcaagtcc atctacatgg ccaagaagcc cgtgaagctg cccggctacc actacgtgga
     1621 ctacaagctc gacatcacct cccacaacga ggactacacc gtggtggagc agtacgagcg
     1681 cgccgaggcc cgccaccacc tgttccagta gctcgagcgg gatcaattcc gccccccccc
     1741 taacgttact ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt
     1801 ttccaccata ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt
     1861 gacgagcatt cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt
     1921 cgtgaaggaa gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct
     1981 ttgcaggcag cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt
     2041 ataagataca cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt
     2101 ggaaagagtc aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa
     2161 ggtaccccat tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta
     2221 gtcgaggtta aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa
     2281 aacacgataa taccatgaac catgggatcg gccattgaac aagatggatt gcacgcaggt
     2341 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     2401 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     2461 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     2521 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     2581 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     2641 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     2701 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     2761 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     2821 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     2881 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     2941 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     3001 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     3061 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagtcga cctgcaggca
     3121 tgcaagcttc aggtagccgg ctaacgttaa caaccggtac ctctagaact atagctagca
     3181 tgcgcaaatt taaagcgctg atatcgataa aataaaagat tttatttagt ctccagaaaa
     3241 aggggggaat gaaagacccc acctgtaggt ttggcaagct agcttaagta acgccatttt
     3301 gcaaggcatg gaaaatacat aactgagaat agagaagttc agatcaaggt taggaacaga
     3361 gagacagcag aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca
     3421 gggccaagaa cagatggtcc ccagatgcgg tcccgccctc agcagtttct agagaaccat
     3481 cagatgtttc cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc
     3541 aatcagttcg cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc
     3601 ccacaacccc tcactcggcg cgccagtcct ccgatagact gcgtcgcccg ggtacccgtg
     3661 tatccaataa accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg
     3721 tctcctctga gtgattgact acccgtcagc gggggtcttt catgggtaac agtttcttga
     3781 agttggagaa caacattctg agggtaggag tcgaatatta agtaatcctg actcaattag
     3841 ccactgtttt gaatccacat actccaatac tcctgaaata gttcattatg gacagcgcag
     3901 aaagagctgg ggagaattgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3961 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4021 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4081 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4141 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4201 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4261 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4321 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     4381 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     4441 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     4501 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     4561 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     4621 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     4681 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     4741 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     4801 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     4861 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     4921 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     4981 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5041 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5101 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5161 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5221 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5281 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     5341 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     5401 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     5461 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     5521 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     5581 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     5641 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     5701 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     5761 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     5821 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     5881 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     5941 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6001 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta
     6061 agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg
     6121 tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt
     6181 cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg
     6241 tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt
     6301 gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg
     6361 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     6421 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     6481 gttttcccag tcacgacgtt gtaaaacgac ggcgcaagga atggtgcatg caaggagatg
     6541 gcgcccaaca gtcccccggc cacggggcct gccaccatac ccacgccgaa acaagcgctc
     6601 atgagcccga agtggcgagc ccgatcttcc ccatcggtga tgtcggcgat ataggcgcca
     6661 gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc gtccggcgta gaggcgatta
     6721 gtccaatttg ttaaagacag gatatcagtg gtccaggctc tagttttgac tcaacaatat
     6781 caccagctga agcctataga gtacgagcca tagataaaat aaaagatttt atttagtctc
     6841 cagaaaaagg ggggaatgaa agaccccacc tgtaggtttg gcaagctagc ttaagtaacg
     6901 ccattttgca aggcatggaa aatacataac tgagaataga gaagttcaga tcaaggttag
     6961 gaacagagag acagcagaat atgggccaaa caggatatct gtggtaagca gttcctgccc
     7021 cggctcaggg ccaagaacag atggtcccca gatgcggtcc cgccctcagc agtttctaga
     7081 gaaccatcag atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga
     7141 actaaccaat cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata
     7201 aaagagccca caacccctca ctcggcgcgc cagtcctccg atagac
//
