LOCUS       sequence-437083-        9290 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses FLPo and mClover3 fluorescent protein with LMCV GP33-43 
            and GP66-77 epitopes embedded in loop of mclover to initiate 
            immunogenic tumor cells in KPFrt mice.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9290)
  TITLE     A dual functional role for T cells enables efficient immunoediting 
            of developing sarcomas
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9290)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: LV-pCMV-FLPo-pGK-mCl-GP33/66
FEATURES             Location/Qualifiers
     source          1..9290
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     448..467
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     LTR             609..789
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     primer_bind     1298..1314
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1299..1315
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1318..1906
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1424..1435
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1833..1853
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(2002..2023)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(2308..2331)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     2799..2820
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(2893..3392)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2941..2960)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     3334..3357
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     CDS             complement(join(3431..4702,4703..4723,4724..4729))
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPo
                     /note="FLPo is a mouse codon-optimized fusion between the 
                     SV40 nuclear localization signal and a thermostable version
                     of the Saccharomyces cerevisiae site-specific recombinase 
                     FLP.  Recombination occurs at FRT sequences."
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     promoter        complement(4808..5011)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        5012..5391
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    5524..5641
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(5939..5983)
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    6168..6401
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    6898..7023
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             7070..7250
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(7268..7466)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        7468..7847
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     8019..8038
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        8113..8217
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8218..8286,8287..9078)
                     /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     complement(8436..8455)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(9249..9290,1..547)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt
       61 tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc
      121 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat
      181 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag
      241 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc
      301 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag
      361 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac
      421 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg
      481 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct
      541 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc
      601 tcacatgctg ctagagattt tccacactga ctaaaagggt ctgagggatc tctagttacc
      661 agagtcacac aacagacggg cacacactac ttgaagcact caaggcaagc tttattgagg
      721 cttaagcagt gggttcccta gttagccaga gagctcccag gctcagatct ggtctaacca
      781 gagagaccca gtacagtccg gatgcagctc tcgggccatg tgatgaaatg ctaggcggct
      841 gtcaaacctc cactctaata cttctctctc cgggtcatcc atcccatgca ggctcacagg
      901 gtgtaacaag cgggtgttct ctccttcatt ggcttcttct accttctctt gctcaactgg
      961 tactagcttg tagcaccatc caaaggtcag tggatatctg atccctggcc ctggtgtgta
     1021 gttctgccaa tcagggaagt agccttgtgt gtggtagatc cacagatcaa ggatatcttg
     1081 tcttcgttgg gagtgaatta gcccttccag tccccccttt tcttttaaaa agtggctaag
     1141 atctacagct gccttgtaag tcattggtct taaaggtacc tgaggtgtga ctggaaaacc
     1201 cacctcctcc tcctcttgtg cttctagcca ggcacaatca gcattggtag ctgctgtatt
     1261 gctacttgtg attgctccat gtttttctag gtctcgatcg aggtcgacgg tatcgatgcg
     1321 gggaggcggc ccaaagggag atccgactcg tctgagggcg aaggcgaaga cgcggaagag
     1381 gccgcagagc cggcagcagg ccgcgggaag gaaggtccgc tggattgagg gccgaaggga
     1441 cgtagcagaa ggacgtcccg cgcagaatcc aggtggcaac acaggcgagc agccaaggaa
     1501 aggacgatga tttccccgac aacaccacgg aattgtcagt gcccaacagc cgagcccctg
     1561 tccagcagcg ggcaaggcag gcggcgatga gttccgccgt ggcaataggg agggggaaag
     1621 cgaaagtccc ggaaaggagc tgacaggtgg tggcaatgcc ccaaccagtg ggggttgcgt
     1681 cagcaaacac agtgcacacc acgccacgtt gcctgacaac gggccacaac tcctcataaa
     1741 gagacagcaa ccaggattta tacaaggagg agaaaatgaa agccatacgg gaagcaatag
     1801 catgatacaa aggcattaaa gcagcgtatc cacatagcgt aaaaggagca acatagttaa
     1861 gaataccagt caatctttca caaattttgt aatccagagg ttgattatcg ggcgcgccgc
     1921 ggtctagaag agtaattcaa ccccaaacaa caacgtttac ttgtacagct cgtccatgcc
     1981 atgtgtaatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt
     2041 ggggtctttg ctcagcttgg actgatggct caggtagtgg ttgtcgggca gcagcacggg
     2101 gccgtcgccg atgggggtgt tctgctggta gtggtcggcg agctgcacgc tgccgtcctc
     2161 aacgttgtgg cggatcttga agttagcctt gatgcagttc ttctgcttgt cgaattctcc
     2221 tgacccagca gcactgcccg cgctgccgtc aaactcaacg gatttaaact gataaactcc
     2281 cttgtaaatg tcagggccgt tcaaaccctt gtcatcgtcg tccttgtagt cgatcccgca
     2341 tgttgcaaag ttgtacacag ctttaaactc gcctgagccg gccgctgatc ctgcagatcc
     2401 agccgtgata tagacgtagt ggctgttgaa gttgtactcc agcttgtgcc ccaggatgtt
     2461 gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg ttcaccaggg tgtcgccctc
     2521 gaacttcacc tcggcgcggg tcttgtaggt accgtcgtcc ttgaaagaga tggtgcgctc
     2581 ctggacgtag ccttcgggca tggcggactt gaagaagtcg tgctgcttca tgtggtcggg
     2641 gtagcggctg aagcaggcca cgccgtagcc gaaggtggtc acgagggtgg gccagggcac
     2701 gggcagcttg ccggtggtgc agatgaactt cagggtcagc ttgccgttgg tggcatcgcc
     2761 ctcgccctcg ccgcggacgc tgaacttgtg gccgtttacg tcgccgtcca gctcgaccag
     2821 gatgggcacc accccggtga acagctcctc gcccttgctc accatggtcc gcttaagctg
     2881 tagcgctaat gtcgggtctc ccggagatga ggaagaggag aacagcgcgg cagacgtgcg
     2941 cttttgaagc gtgcagaatg ccgggcctcc ggaggacctt cgggcgcccg ccccgcccct
     3001 gagcccgccc ctgagcccgc ccccggaccc accccttccc agcctctgag cccagaaagc
     3061 gaaggagcaa agctgctatt ggccgctgcc ccaaaggcct acccgcttcc attgctcagc
     3121 ggtgctgtcc atctgcacga gactagtgag acgtgctact tccatttgtc acgtcctgca
     3181 cgacgcgagc tgcggggcgg gggggaactt cctgactagg ggaggagtag aaggtggcgc
     3241 gaaggggcca ccaaagaacg gagccggttg gcgcctaccg gtggatgtgg aatgtgtgcg
     3301 aggccagagg ccacttgtgt agcgccaagt gcccagcggg gctgctaaag cgcatgctcc
     3361 agactgcctt gggaaaagcg cctcccctac ccggtagaat aggcctcggg attcctagga
     3421 acagcggttt tcagatccgc ctgttgatgt agctgctcag gtagtccagc acctcctggc
     3481 tgatgatgcc gttccaggcg gggtatctga tgctgccctc ggcgctgccc ttcagctgct
     3541 cgatgtgctg ccactcctcg atggggttgg tctcgtcctt cagggcgatc atctccttgc
     3601 tgatggggtc gtaggcgtag tacctggaca ccagggcgaa gtagtggtcg gggatggcgg
     3661 tgatctggtg ggtgtaggtg gtcctggcca cggcggaggc cctcttgtcg ctccagttgc
     3721 ccaccacgtt tgtcagctcg gtcaggccct tcatgctcag aaagctggtc atcaggtgcc
     3781 tgccgatgtg gctcttaggg ccgttcttga tagcgaagat ggggtagggg gcgttcttct
     3841 tcagggcctt gttgtagctg cgcaccaggt tgtccttcag cagctggtac tcctgcttgt
     3901 tgctgctgct gttgccggtc ctgttcactc tcttcagcac gggctcgctg ttcctcagga
     3961 actcgtccag gtacaccagg gggtcgatcc tgcctctggc gctgaaaaag tagatgtgcc
     4021 tggacacgct tgtcttggtc tcggtcacca ggcactgaat gatcacgccc aggtacttgt
     4081 tctgcaccag cttgaagctc ttggggtcca cgttcttgat gtcgctgaac ctgccgcagt
     4141 tgatgaatgt ggccaggaac aggaactggt acagggtctt ggtcttggtg aacctgctgg
     4201 tgtactcgaa gctgttcagg atcttctcgg tgatctccca gatgctctcg ccctcggaca
     4261 gcagggcctt cagcatcttc ttgctgtggc tgttgccctt gtcggcctcc tcgctgctct
     4321 cgaactgcag ctgcaggctg gacacgatgt cggtgatgtc gctctggtgc ttctggccgt
     4381 tgtaagggat gatggtgaac tcccaggcgg ggatcagctt cttcaggctg gcctccagga
     4441 tggtggcctt ctgggtcttg tacttgaact gcaggctctt gttcacgatg tcgaagctca
     4501 ggctgttgct gatgatggtg ttgtagctca tgaaggtggc cctcttgatg gcggtgccgt
     4561 tgtgggtgat catccagcac aggtaggtca gctcggcggc acagctggcg atcttctcgc
     4621 cgctgggcct ctcgaatctc tccacgaact gccgcaccag caccttgggg ggggtcttgc
     4681 acaggatgtc gaactggctc atcaccttcc tcttcttctt aggagccatg gtaaacgatc
     4741 gcacagctag cgttcgagtt agaatttcga taagccagta agcagtgggt tctctagtta
     4801 gccagagagc tctgcttata tagacctccc accgtacacg cctaccgccc atttgcgtca
     4861 atggggcgga gttgttacga cattttggaa agtcccgttg attttggtgc caaaacaaac
     4921 tcccattgac gtcaatgggg tggagacttg gaaatccccg tgagtcaaac cgctatccac
     4981 gcccattgat gtactgccaa aaccgcatca ccatggtaat agcgatgact aatacgtaga
     5041 tgtactgcca agtaggaaag tcccataagg tcatgtactg ggcataatgc caggcgggcc
     5101 atttaccgtc attgacgtca atagggggcg tacttggcat atgatacact tgatgtactg
     5161 ccaagtgggc agtttaccgt aaatactcca cccattgacg tcaatggaaa gtccctattg
     5221 gcgttactat gggaacatac gtcattattg acgtcaatgg gcgggggtcg ttgggcggtc
     5281 agccaggcgg gccatttacc gtaagttatg taacgcggaa ctccatatat gggctatgaa
     5341 ctaatgaccc cgtaattgat tactattaat aactagtcaa taatcaatgt caacgcgtat
     5401 atctggcccg tacatcgcga agcagcgcaa aaaaccagct ttacactccc tcacactgat
     5461 cactagtcga cccgggcccg gtactaacca aactggatct ctgctgtccc tgtaataaac
     5521 ccgaaaattt tgaatttttg taatttgttt ttgtaattct ttagtttgta tgtctgttgc
     5581 tattatgtct actattcttt cccctgcact gtacccccca atcccccctt ttcttttaaa
     5641 attgtggatg aatactgcca tttgtctgca gaattggcgc acgcagtgcc gatccgttca
     5701 ctaatcgaat ggatctgtct ctgtctctct ctccaccttc ttcttctatt ccttcgggcc
     5761 tgtcgggtcc cctcggggtt gggaggtggg tctgaaacga taatggtgaa tatccctgcc
     5821 taactctatt cactatagaa agtacagcaa aaactattct taaacctacc aagcctccta
     5881 ctatcattat gaataatttt atataccaca gccaatttgt tatgttaaac caattccaca
     5941 aacttgccca tttatctaat tccaataatt cttgttcatt cttttcttgc tggttttgcg
     6001 attcttcaat taaggagtgt attaagcttg tgtaattgtt aatttctctg tcccactcca
     6061 tccaggtcgt gtgattccaa atctgttcca gagatttatt actccaacta gcattccaag
     6121 gcacagcagt ggtgcaaatg agttttccag agcaacccca aatccccagg agctgttgat
     6181 cctttaggta tctttccaca gccaggattc ttgcctggag ctgcttgatg ccccagactg
     6241 tgagttgcaa cagatgctgt tgcgcctcaa tagccctcag caaattgttc tgctgctgca
     6301 ctataccaga caataattgt ctggcctgta ccgtcagcgt cattgacgct gcgcccatag
     6361 tgcttcctgc tgctcccaag aacccaagga acaaagctcc tattcccact gctctttttt
     6421 ctctctgcac cactcttctc tttgccttgg tgggtgctac tcctaatggt tcaattttta
     6481 ctactttata tttatataat tcacttctcc aattgtccct catatctcct cctccaggtc
     6541 tgaagatcag cggccggccg cttgctgtgc ggtggtctta cttttgtttt gctcttcctc
     6601 tatcttgtct aaagcttcct tggtgtcttt tatctctatc ctttgatgca cacaatagag
     6661 ggttgctact gtattatata atgatctaag ttcttctgat cctgtctgaa gggatggttg
     6721 tagctgtccc agtatttgtc tacagccttc tgatgtttct aacaggccag gattaactgc
     6781 gaatcgttct agctccctgc ttgcccatac tatatgtttt aatttatatt ttttctttcc
     6841 ccctggcctt aaccgaattt tttcccatcg cgatctaatt ctcccccgct taatactgac
     6901 gctctcgcac ccatctctct ccttctagcc tccgctagtc aaaatttttg gcgtactcac
     6961 cagtcgccgc ccctcgcctc ttgccgtgcg cgcttcagca agccgagtcc tgcgtcgaga
     7021 gagctcctct ggtttccctt tcgctttcaa gtccctgttc gggcgccact gctagagatt
     7081 ttccacactg actaaaaggg tctgagggat ctctagttac cagagtcaca caacagacgg
     7141 gcacacacta cttgaagcac tcaaggcaag ctttattgag gcttaagcag tgggttccct
     7201 agttagccag agagctccca ggctcagatc tggtctaacc agagagaccc agtacaggca
     7261 aaacgcgctg cttatataga cctcccaccg tacacgccta ccgcccattt gcgtcaatgg
     7321 ggcggagttg ttacgacatt ttggaaagtc ccgttgattt tggtgccaaa acaaactccc
     7381 attgacgtca atggggtgga gacttggaaa tccccgtgag tcaaaccgct atccacgccc
     7441 attgatgtac tgccaaaacc gcatcaccat ggtaatagcg atgactaata cgtagatgta
     7501 ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt
     7561 accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa
     7621 gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt
     7681 tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc
     7741 aggcgggcca tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa
     7801 tgaccccgta attgattact attaataact agtcaataat caatgtcaac gcgtatatct
     7861 ggcccgtaca tcgcgaagca gcgcaaaacg cctaacccta agcagattct tcatgcaatt
     7921 gtcggtcaag ccttgccttg ttgtagctta aattttgctc gcgcactact cagcgacctc
     7981 caacacacaa gcagggagca gatactggct taactatgcg gcatcagagc agattgtact
     8041 gagagtgcac cataggggat cgggagatct cccgatccgt cgacgtcagg tggcactttt
     8101 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     8161 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     8221 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     8281 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     8341 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     8401 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     8461 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     8521 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     8581 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     8641 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     8701 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     8761 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     8821 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     8881 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     8941 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     9001 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     9061 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     9121 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     9181 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     9241 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac
//
