LOCUS       sequence-306230-        7273 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral (MSCV) expression of LSSmOrange fluorescent protein .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7273)
  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 7273)
  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-LO
FEATURES             Location/Qualifiers
     source          1..7273
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    64..405
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             472..888
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     818..840
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     856..872
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      897..906
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             903..1613
                     /codon_start=1
                     /product="monomeric orange fluorescent protein with a large
                     Stokes shift (Shcherbakova et al., 2012)"
                     /label=LSSmOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALK
                     GEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     1316..1335
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     misc_feature    1645..2194
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1789..1806)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2016..2035
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             2204..3007
                     /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(2267..2286)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2877..2896
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     LTR             3151..3665
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    3827..3843
                     /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(3851..3857,3858..3875,3876..3881))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3896..3917
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4034..4051)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4205..4793)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4285..4304)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4964..5755,5756..5824))
                     /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     5587..5606
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5825..5929)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5997..6015
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6053..6075)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6175..6194
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6388..6410
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6538..6557
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             6757..7273
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tggaggttcc accgagattt ggagacccct gcccagggac caccgacccc cccgccggga
       61 ggtaagctgg ccagcggtcg tttcgtgtct gtctctgtct ttgtgcgtgt ttgtgccggc
      121 atctaatgtt tgcgcctgcg tctgtactag ttagctaact agctctgtat ctggcggacc
      181 cgtggtggaa ctgacgagtt ctgaacaccc ggccgcaacc ctgggagacg tcccagggac
      241 tttgggggcc gtttttgtgg cccgacctga ggaagggagt cgatgtggaa tccgaccccg
      301 tcaggatatg tggttctggt aggagacgag aacctaaaac agttcccgcc tccgtctgaa
      361 tttttgcttt cggtttggaa ccgaagccgc gcgtcttgtc tgctgcagcg ctgcagcatc
      421 gttctgtgtt gtctctgtct gactgtgttt ctgtatttgt ctgaaaatta gggccagact
      481 gttaccactc ccttaagttt gaccttaggt cactggaaag atgtcgagcg gatcgctcac
      541 aaccagtcgg tagatgtcaa gaagagacgt tgggttacct tctgctctgc agaatggcca
      601 acctttaacg tcggatggcc gcgagacggc acctttaacc gagacctcat cacccaggtt
      661 aagatcaagg tcttttcacc tggcccgcat ggacacccag accaggtccc ctacatcgtg
      721 acctgggaag ccttggcttt tgacccccct ccctgggtca agccctttgt acaccctaag
      781 cctccgcctc ctcttcctcc atccgccccg tctctccccc ttgaacctcc tcgttcgacc
      841 ccgcctcgat cctcccttta tccagccctc actccttctc taggcgccgg aattcagcca
      901 ccatggtgag caagggcgag gagaataaca tggccatcat caaggagttc atgcgcttca
      961 aggtgcgcat ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg
     1021 gccgccccta cgagggcttt cagaccgtta agctgaaggt gaccaagggt ggccccctgc
     1081 ccttcgcctg ggacatcttg tcccctcagt tcacctacgg ctccaaggcc tacgtgaagc
     1141 accccgccga catccccgac tacctcaagc tgtccttccc cgagggcttc aagtgggagc
     1201 gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac tcaggactcc tccctgcagg
     1261 acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg
     1321 taatgcagaa gaagaccatg ggcatggagg cctcctccga gcggatgtac cccgaggacg
     1381 gcgccctgaa gggcgaggac aagctcaggc tgaagctgaa ggacggcggc cactacacct
     1441 ccgaggtcaa gaccacctac aaggccaaga agcccgtgca gttgcccggc gcctacatcg
     1501 tcgacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg
     1561 aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag taactcgagc
     1621 gggatcaatt ccgccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt
     1681 gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc
     1741 ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag
     1801 gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac
     1861 aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc
     1921 tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc
     1981 acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca
     2041 aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt
     2101 gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg
     2161 gggacgtggt tttcctttga aaaacacgat aataccatga accatgggat cggccattga
     2221 acaagatgga ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga
     2281 ctgggcacaa cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg
     2341 gcgcccggtt ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga
     2401 ggcagcgcgg ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt
     2461 tgtcactgaa gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct
     2521 gtcatctcac cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct
     2581 gcatacgctt gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg
     2641 agcacgtact cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca
     2701 ggggctcgcg ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga
     2761 tctcgtcgtg acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt
     2821 ttctggattc atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt
     2881 ggctacccgt gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct
     2941 ttacggtatc gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt
     3001 cttctgagtc gacctgcagg catgcaagct tcaggtagcc ggctaacgtt aacaaccggt
     3061 acctctagaa ctatagctag catgcgcaaa tttaaagcgc tgatatcgat aaaataaaag
     3121 attttattta gtctccagaa aaagggggga atgaaagacc ccacctgtag gtttggcaag
     3181 ctagcttaag taacgccatt ttgcaaggca tggaaaatac ataactgaga atagagaagt
     3241 tcagatcaag gttaggaaca gagagacagc agaatatggg ccaaacagga tatctgtggt
     3301 aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc ggtcccgccc
     3361 tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc
     3421 ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc
     3481 tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc ctccgataga
     3541 ctgcgtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg
     3601 gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct
     3661 ttcatgggta acagtttctt gaagttggag aacaacattc tgagggtagg agtcgaatat
     3721 taagtaatcc tgactcaatt agccactgtt ttgaatccac atactccaat actcctgaaa
     3781 tagttcatta tggacagcgc agaaagagct ggggagaatt gtgaaattgt tatccgctca
     3841 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     3901 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     3961 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4021 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4081 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4141 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4201 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4261 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4321 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4381 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4441 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4501 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4561 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     4621 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     4681 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     4741 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     4801 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     4861 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     4921 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     4981 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5041 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5101 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5161 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5221 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5281 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5341 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5401 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5461 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5521 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     5581 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     5641 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     5701 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     5761 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     5821 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     5881 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     5941 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     6001 ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa aacctctgac
     6061 acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag
     6121 cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac tatgcggcat
     6181 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
     6241 ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt tgggaagggc
     6301 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc
     6361 gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggcgcaag
     6421 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc ctgccaccat
     6481 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt ccccatcggt
     6541 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc cggccacgat
     6601 gcgtccggcg tagaggcgat tagtccaatt tgttaaagac aggatatcag tggtccaggc
     6661 tctagttttg actcaacaat atcaccagct gaagcctata gagtacgagc catagataaa
     6721 ataaaagatt ttatttagtc tccagaaaaa ggggggaatg aaagacccca cctgtaggtt
     6781 tggcaagcta gcttaagtaa cgccattttg caaggcatgg aaaatacata actgagaata
     6841 gagaagttca gatcaaggtt aggaacagag agacagcaga atatgggcca aacaggatat
     6901 ctgtggtaag cagttcctgc cccggctcag ggccaagaac agatggtccc cagatgcggt
     6961 cccgccctca gcagtttcta gagaaccatc agatgtttcc agggtgcccc aaggacctga
     7021 aatgaccctg tgccttattt gaactaacca atcagttcgc ttctcgcttc tgttcgcgcg
     7081 cttctgctcc ccgagctcaa taaaagagcc cacaacccct cactcggcgc gccagtcctc
     7141 cgatagactg cgtcgcccgg gtacccgtat tcccaataaa gcctcttgct gtttgcatcc
     7201 gaatcgtgga ctcgctgatc cttgggaggg tctcctcaga ttgattgact gcccacctcg
     7261 ggggtctttc att
//
