LOCUS       sequence-319731-        5853 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 5853)
  AUTHORS   Landry LG, Mann SE, Anderson AM, Nakayama M
  TITLE     Multiplex T-cell Stimulation Assay Utilizing a T-cell Activation 
            Reporter-based Detection System.
  JOURNAL   Bio Protoc. 2021 Jan 20;11(2):e3883. doi: 10.21769/BioProtoc.3883. 
            eCollection 2021 Jan 20.
  PUBMED    33732772
REFERENCE   2  (bases 1 to 5853)
  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: "Bio 
            Protoc."; date: "2021-01-20"; volume: "11(2):e3883. doi"; pages: " 
            10.21769/BioProtoc.3883. eCollection 2021 Jan 20"
COMMENT     Sequence Label: pMSCVII-E2Crimson
FEATURES             Location/Qualifiers
     source          1..5853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..517
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
     misc_feature    581..922
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             989..1405
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1335..1357
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1373..1389
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1420..1429
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1426..2103
                     /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     1824..1843
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     LTR             2248..2762
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    2924..2940
                     /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(2948..2954,2955..2972,2973..2978))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2993..3014
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3131..3148)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3302..3890)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3382..3401)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4061..4852,4853..4921))
                     /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     4684..4703
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4922..5026)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5094..5112
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5150..5172)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5272..5291
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5485..5507
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     5635..5654
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
       61 atggaaaata cataactgag aatagagaag ttcagatcaa ggttaggaac agagagacag
      121 cagaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
      181 gaacagatgg tccccagatg cggtcccgcc ctcagcagtt tctagagaac catcagatgt
      241 ttccagggtg ccccaaggac ctgaaatgac cctgtgcctt atttgaacta accaatcagt
      301 tcgcttctcg cttctgttcg cgcgcttctg ctccccgagc tcaataaaag agcccacaac
      361 ccctcactcg gcgcgccagt cctccgatag actgcgtcgc ccgggtaccc gtattcccaa
      421 taaagcctct tgctgtttgc atccgaatcg tggactcgct gatccttggg agggtctcct
      481 cagattgatt gactgcccac ctcgggggtc tttcatttgg aggttccacc gagatttgga
      541 gacccctgcc cagggaccac cgaccccccc gccgggaggt aagctggcca gcggtcgttt
      601 cgtgtctgtc tctgtctttg tgcgtgtttg tgccggcatc taatgtttgc gcctgcgtct
      661 gtactagtta gctaactagc tctgtatctg gcggacccgt ggtggaactg acgagttctg
      721 aacacccggc cgcaaccctg ggagacgtcc cagggacttt gggggccgtt tttgtggccc
      781 gacctgagga agggagtcga tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg
      841 agacgagaac ctaaaacagt tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg
      901 aagccgcgcg tcttgtctgc tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac
      961 tgtgtttctg tatttgtctg aaaattaggg ccagactgtt accactccct taagtttgac
     1021 cttaggtcac tggaaagatg tcgagcggat cgctcacaac cagtcggtag atgtcaagaa
     1081 gagacgttgg gttaccttct gctctgcaga atggccaacc tttaacgtcg gatggccgcg
     1141 agacggcacc tttaaccgag acctcatcac ccaggttaag atcaaggtct tttcacctgg
     1201 cccgcatgga cacccagacc aggtccccta catcgtgacc tgggaagcct tggcttttga
     1261 cccccctccc tgggtcaagc cctttgtaca ccctaagcct ccgcctcctc ttcctccatc
     1321 cgccccgtct ctcccccttg aacctcctcg ttcgaccccg cctcgatcct ccctttatcc
     1381 agccctcact ccttctctag gcgccggaat tcagatctcg ccaccatgga tagcactgag
     1441 aacgtcatca agcccttcat gcgcttcaag gtgcacatgg agggctccgt gaacggccac
     1501 gagttcgaga tcgagggcgt gggcgagggc aagccctacg agggcaccca gaccgccaag
     1561 ctgcaagtga ccaagggcgg ccccctgccc ttcgcctggg acatcctgtc cccccagttc
     1621 ttctacggct ccaaggcgta catcaagcac cccgccgaca tccccgacta cctcaagcag
     1681 tccttccccg agggcttcaa gtgggagcgc gtgatgaact tcgaggacgg cggcgtggtg
     1741 accgtgaccc aggactcctc cctgcaggac ggcaccctca tctaccacgt gaagttcatc
     1801 ggcgtgaact tcccctccga cggccccgta atgcagaaga agactctggg ctgggagccc
     1861 tccactgagc gcaactaccc ccgcgacggc gtgctgaagg gcgagaacca catggcgctg
     1921 aagctgaagg gcggcggcca ctacctgtgt gagttcaagt ccatctacat ggccaagaag
     1981 cccgtgaagc tgcccggcta ccactacgtg gactacaagc tcgacatcac ctcccacaac
     2041 gaggactaca ccgtggtgga gcagtacgag cgcgccgagg cccgccacca cctgttccag
     2101 tagctcgagc ctgcaggcat gcaagcttca ggtagccggc taacgttaac aaccggtacc
     2161 tctagaacta tagctagcat gcgcaaattt aaagcgctga tatcgataaa ataaaagatt
     2221 ttatttagtc tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta
     2281 gcttaagtaa cgccattttg caaggcatgg aaaatacata actgagaata gagaagttca
     2341 gatcaaggtt aggaacagag agacagcaga atatgggcca aacaggatat ctgtggtaag
     2401 cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt cccgccctca
     2461 gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga aatgaccctg
     2521 tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg cttctgctcc
     2581 ccgagctcaa taaaagagcc cacaacccct cactcggcgc gccagtcctc cgatagactg
     2641 cgtcgcccgg gtacccgtgt atccaataaa ccctcttgca gttgcatccg acttgtggtc
     2701 tcgctgttcc ttgggagggt ctcctctgag tgattgacta cccgtcagcg ggggtctttc
     2761 atgggtaaca gtttcttgaa gttggagaac aacattctga gggtaggagt cgaatattaa
     2821 gtaatcctga ctcaattagc cactgttttg aatccacata ctccaatact cctgaaatag
     2881 ttcattatgg acagcgcaga aagagctggg gagaattgtg aaattgttat ccgctcacaa
     2941 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     3001 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     3061 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3121 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3181 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3241 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3301 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3361 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3421 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3481 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3541 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3601 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3661 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3721 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3781 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3841 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3901 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3961 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4021 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4081 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4141 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4201 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4261 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4321 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4381 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4441 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4501 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4561 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4621 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4681 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4741 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4801 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4861 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4921 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4981 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5041 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5101 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     5161 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     5221 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     5281 agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga
     5341 gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     5401 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     5461 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gcgcaaggaa
     5521 tggtgcatgc aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc
     5581 cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat
     5641 gtcggcgata taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg
     5701 tccggcgtag aggcgattag tccaatttgt taaagacagg atatcagtgg tccaggctct
     5761 agttttgact caacaatatc accagctgaa gcctatagag tacgagccat agataaaata
     5821 aaagatttta tttagtctcc agaaaaaggg ggg
//
