LOCUS       Exported                8312 bp ds-DNA     linear   SYN 27-MAY-2021
DEFINITION  PresentER minigene encoding H2-Kb ligand MSIIFFLPL (GFP).
ACCESSION   .
VERSION     .
KEYWORDS    PresentER-MSIIFFLPL (GFP)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8312)
  AUTHORS   Gejman RS, Chang AY, Jones HF, DiKun K, Hakimi AA, Schietinger A, 
            Scheinberg DA
  TITLE     Rejection of immunogenic tumor clones is limited by clonal fraction.
  JOURNAL   Elife. 2018 Nov 30;7. pii: 41090. doi: 10.7554/eLife.41090.
  PUBMED    30499773
REFERENCE   2  (bases 1 to 8312)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 27, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8312
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     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"
     ncRNA           1445..1538
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           1968..2080
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     promoter        2094..2593
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2129..2152)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2526..2545
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2614..3213
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2614..2633)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3110..3130
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    3291..3843
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3435..3452)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3662..3681
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3855..4574
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3900..3921)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4161..4180)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4508..4529
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     LTR             4676..5190
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    5383..5399
                     /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(5407..5437)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5452..5473
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5590..5607)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5761..6349)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5841..5860)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6520..7380)
                     /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     7143..7162
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7381..7485)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7553..7571
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(7609..7631)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     7731..7750
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7944..7966
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     8094..8113
                     /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 tagatctctc gactagggat aacagggtaa
     1441 ttgtttgaat gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt
     1501 gctgggatta cttcttcagg ttaacccaac agaaggctcg aggtgactgg agttcagacg
     1561 tgtgctcttc cgatcgccac catgcctaat catcagtccg ggtcacctac cggcagttca
     1621 gacctgctcc ttgatggcaa gaaacaacga gcccatctgg cgctgaggag aaaacggcga
     1681 cgggaaatgc gcaagattaa ccgaaaggtg agaagaatga atctcgcacc cattaaagaa
     1741 aaaacagcct ggcagcacct gcaagctctg atcttcgagg cggaagaagt gttgaagact
     1801 tctcaaactc cacagacctc cctcacgctg tttctagcac tcttggccgt attggccccg
     1861 ccacctgtga gcgggatgag catcatcttc ttcctgcccc tgtaaggcca aacaggccaa
     1921 agatcggaag agcgtcgtgt agggaaagag tgtaccgtat cattgaattc aaggggctac
     1981 tttaggagca attatcttgt ttactaaaac tgaatacctt gctatctctt tgatacattt
     2041 ttacaaagct gaattaaaat ggtataaatt aaatcacttt tttcaattct accgggtagg
     2101 ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg
     2161 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc
     2221 ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt
     2281 cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac
     2341 tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag
     2401 cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc
     2461 cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg
     2521 cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc
     2581 tccgggcctt tcgacctgca gcccaagctt accatgaccg agtacaagcc cacggtgcgc
     2641 ctcgccaccc gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac
     2701 taccccgcca cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg
     2761 caagaactct tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac
     2821 ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc
     2881 gagatcggcc cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg
     2941 gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc
     3001 gtctcgcccg accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag
     3061 gcggccgagc gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc
     3121 ttctacgagc ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc
     3181 acctggtgca tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg
     3241 accgaaagga gcgcacgacc ccatgcatcc aattccgccc ccccccccta acgttactgg
     3301 ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt ccaccatatt
     3361 gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga cgagcattcc
     3421 taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc
     3481 agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt gcaggcagcg
     3541 gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat aagatacacc
     3601 tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg aaagagtcaa
     3661 atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg taccccattg
     3721 tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa
     3781 aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa cacgatgata
     3841 atatggccac aaccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     3901 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtctggc gagggcgagg
     3961 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     4021 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     4081 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     4141 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     4201 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     4261 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     4321 acaagcagaa gaacggcatc aaggcgaact tcaagatccg ccacaacatc gaggacggca
     4381 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     4441 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     4501 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     4561 agctgtacaa gtaatgaatt aattaagaat tatcaagctt atcgataccg tcgacctgca
     4621 gccaagctta tcgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa
     4681 agaccccacc tgtaggtttg gcaagctagc ttaagtaacg ccattttgca aggcatggaa
     4741 aatacataac tgagaataga gaagttcaga tcaaggttag gaacagagag acagcagaat
     4801 atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg ccaagaacag
     4861 atggtcccca gatgcggtcc cgccctcagc agtttctaga gaaccatcag atgtttccag
     4921 ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat cagttcgctt
     4981 ctcgcttctg ttcgcgcgct tctgctcccc gagctcaata aaagagccca caacccctca
     5041 ctcggcgcgc cagtcctccg atagactgcg tcgcccgggt acccgtgtat ccaataaacc
     5101 ctcttgcagt tgcatccgac ttgtggtctc gctgttcctt gggagggtct cctctgagtg
     5161 attgactacc cgtcagcggg ggtctttcat gggtaacagt ttcttgaagt tggagaacaa
     5221 cattctgagg gtaggagtcg aatattaagt aatcctgact caattagcca ctgttttgaa
     5281 tccacatact ccaatactcc tgaaatagtt cattatggac agcgcagaaa gagctgggga
     5341 gcattatgga cagcgcagaa agagctgggg agaattgtga aattgttatc cgctcacaat
     5401 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     5461 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     5521 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     5581 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     5641 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     5701 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     5761 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     5821 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     5881 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     5941 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6001 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6061 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6121 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     6181 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     6241 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     6301 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     6361 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     6421 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     6481 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     6541 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     6601 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     6661 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     6721 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     6781 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     6841 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     6901 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     6961 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7021 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7081 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7141 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     7201 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     7261 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     7321 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     7381 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     7441 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     7501 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     7561 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     7621 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     7681 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     7741 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     7801 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     7861 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     7921 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg cgcaaggaat
     7981 ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc
     8041 acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg
     8101 tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt
     8161 ccggcgtaga ggcgattagt ccaatttgtt aaagacagga tatcagtggt ccaggctcta
     8221 gttttgactc aacaatatca ccagctgaag cctatagagt acgagccata gataaaataa
     8281 aagattttat ttagtctcca gaaaaagggg gg
//
