LOCUS       sequence-437085-        9078 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses a lox-p flanked FLPo and mClover3 fluorescent protein with
            LMCV GP33-43 and GP66-77 epitopes embedded in loop of mclover for 
            initiating and removing immunogenic tumor cells in KPFrt mice.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9078)
  TITLE     A dual functional role for T cells enables efficient immunoediting 
            of developing sarcomas
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 9078)
  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            "Unpublished"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: pLox-LV-pCMV-FLPo-pGK-mCl-GP33/66
FEATURES             Location/Qualifiers
     source          1..9078
                     /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"
     protein_bind    809..842
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1086..1102
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1087..1103
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    1106..1694
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1212..1223
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1621..1641
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     primer_bind     complement(1790..1811)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(2096..2119)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     2587..2608
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     promoter        complement(2681..3180)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2729..2748)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     3122..3145
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     CDS             complement(join(3219..4490,4491..4511,4512..4517))
                     /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(4596..4799)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        4800..5179
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    5312..5429
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(5727..5771)
                     /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    5956..6189
                     /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    6686..6811
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             6858..7038
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(7056..7254)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     enhancer        7256..7635
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     7807..7826
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        7901..8005
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8006..8074,8075..8866)
                     /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(8224..8243)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(9037..9078,1..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 gtacaagcaa aaagcagcat aacttcgtat aatgtatgct atacgaagtt
      841 attcccatgg tgaattgatc ccatcttgtc ttcgttggga gtgaattagc ccttccagtc
      901 cccccttttc ttttaaaaag tggctaagat ctacagctgc cttgtaagtc attggtctta
      961 aaggtacctg aggtgtgact ggaaaaccca cctcctcctc ctcttgtgct tctagccagg
     1021 cacaatcagc attggtagct gctgtattgc tacttgtgat tgctccatgt ttttctaggt
     1081 ctcgatcgag gtcgacggta tcgatgcggg gaggcggccc aaagggagat ccgactcgtc
     1141 tgagggcgaa ggcgaagacg cggaagaggc cgcagagccg gcagcaggcc gcgggaagga
     1201 aggtccgctg gattgagggc cgaagggacg tagcagaagg acgtcccgcg cagaatccag
     1261 gtggcaacac aggcgagcag ccaaggaaag gacgatgatt tccccgacaa caccacggaa
     1321 ttgtcagtgc ccaacagccg agcccctgtc cagcagcggg caaggcaggc ggcgatgagt
     1381 tccgccgtgg caatagggag ggggaaagcg aaagtcccgg aaaggagctg acaggtggtg
     1441 gcaatgcccc aaccagtggg ggttgcgtca gcaaacacag tgcacaccac gccacgttgc
     1501 ctgacaacgg gccacaactc ctcataaaga gacagcaacc aggatttata caaggaggag
     1561 aaaatgaaag ccatacggga agcaatagca tgatacaaag gcattaaagc agcgtatcca
     1621 catagcgtaa aaggagcaac atagttaaga ataccagtca atctttcaca aattttgtaa
     1681 tccagaggtt gattatcggg cgcgccgcgg tctagaagag taattcaacc ccaaacaaca
     1741 acgtttactt gtacagctcg tccatgccat gtgtaatccc ggcggcggtc acgaactcca
     1801 gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct cagcttggac tgatggctca
     1861 ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat gggggtgttc tgctggtagt
     1921 ggtcggcgag ctgcacgctg ccgtcctcaa cgttgtggcg gatcttgaag ttagccttga
     1981 tgcagttctt ctgcttgtcg aattctcctg acccagcagc actgcccgcg ctgccgtcaa
     2041 actcaacgga tttaaactga taaactccct tgtaaatgtc agggccgttc aaacccttgt
     2101 catcgtcgtc cttgtagtcg atcccgcatg ttgcaaagtt gtacacagct ttaaactcgc
     2161 ctgagccggc cgctgatcct gcagatccag ccgtgatata gacgtagtgg ctgttgaagt
     2221 tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct
     2281 cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtaggtac
     2341 cgtcgtcctt gaaagagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga
     2401 agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcaggccacg ccgtagccga
     2461 aggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca
     2521 gggtcagctt gccgttggtg gcatcgccct cgccctcgcc gcggacgctg aacttgtggc
     2581 cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc
     2641 ccttgctcac catggtccgc ttaagctgta gcgctaatgt cgggtctccc ggagatgagg
     2701 aagaggagaa cagcgcggca gacgtgcgct tttgaagcgt gcagaatgcc gggcctccgg
     2761 aggaccttcg ggcgcccgcc ccgcccctga gcccgcccct gagcccgccc ccggacccac
     2821 cccttcccag cctctgagcc cagaaagcga aggagcaaag ctgctattgg ccgctgcccc
     2881 aaaggcctac ccgcttccat tgctcagcgg tgctgtccat ctgcacgaga ctagtgagac
     2941 gtgctacttc catttgtcac gtcctgcacg acgcgagctg cggggcgggg gggaacttcc
     3001 tgactagggg aggagtagaa ggtggcgcga aggggccacc aaagaacgga gccggttggc
     3061 gcctaccggt ggatgtggaa tgtgtgcgag gccagaggcc acttgtgtag cgccaagtgc
     3121 ccagcggggc tgctaaagcg catgctccag actgccttgg gaaaagcgcc tcccctaccc
     3181 ggtagaatag gcctcgggat tcctaggaac agcggttttc agatccgcct gttgatgtag
     3241 ctgctcaggt agtccagcac ctcctggctg atgatgccgt tccaggcggg gtatctgatg
     3301 ctgccctcgg cgctgccctt cagctgctcg atgtgctgcc actcctcgat ggggttggtc
     3361 tcgtccttca gggcgatcat ctccttgctg atggggtcgt aggcgtagta cctggacacc
     3421 agggcgaagt agtggtcggg gatggcggtg atctggtggg tgtaggtggt cctggccacg
     3481 gcggaggccc tcttgtcgct ccagttgccc accacgtttg tcagctcggt caggcccttc
     3541 atgctcagaa agctggtcat caggtgcctg ccgatgtggc tcttagggcc gttcttgata
     3601 gcgaagatgg ggtagggggc gttcttcttc agggccttgt tgtagctgcg caccaggttg
     3661 tccttcagca gctggtactc ctgcttgttg ctgctgctgt tgccggtcct gttcactctc
     3721 ttcagcacgg gctcgctgtt cctcaggaac tcgtccaggt acaccagggg gtcgatcctg
     3781 cctctggcgc tgaaaaagta gatgtgcctg gacacgcttg tcttggtctc ggtcaccagg
     3841 cactgaatga tcacgcccag gtacttgttc tgcaccagct tgaagctctt ggggtccacg
     3901 ttcttgatgt cgctgaacct gccgcagttg atgaatgtgg ccaggaacag gaactggtac
     3961 agggtcttgg tcttggtgaa cctgctggtg tactcgaagc tgttcaggat cttctcggtg
     4021 atctcccaga tgctctcgcc ctcggacagc agggccttca gcatcttctt gctgtggctg
     4081 ttgcccttgt cggcctcctc gctgctctcg aactgcagct gcaggctgga cacgatgtcg
     4141 gtgatgtcgc tctggtgctt ctggccgttg taagggatga tggtgaactc ccaggcgggg
     4201 atcagcttct tcaggctggc ctccaggatg gtggccttct gggtcttgta cttgaactgc
     4261 aggctcttgt tcacgatgtc gaagctcagg ctgttgctga tgatggtgtt gtagctcatg
     4321 aaggtggccc tcttgatggc ggtgccgttg tgggtgatca tccagcacag gtaggtcagc
     4381 tcggcggcac agctggcgat cttctcgccg ctgggcctct cgaatctctc cacgaactgc
     4441 cgcaccagca ccttgggggg ggtcttgcac aggatgtcga actggctcat caccttcctc
     4501 ttcttcttag gagccatggt aaacgatcgc acagctagcg ttcgagttag aatttcgata
     4561 agccagtaag cagtgggttc tctagttagc cagagagctc tgcttatata gacctcccac
     4621 cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag
     4681 tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg gagacttgga
     4741 aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa ccgcatcacc
     4801 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
     4861 atgtactggg cataatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
     4921 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
     4981 cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac
     5041 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
     5101 acgcggaact ccatatatgg gctatgaact aatgaccccg taattgatta ctattaataa
     5161 ctagtcaata atcaatgtca acgcgtatat ctggcccgta catcgcgaag cagcgcaaaa
     5221 aaccagcttt acactccctc acactgatca ctagtcgacc cgggcccggt actaaccaaa
     5281 ctggatctct gctgtccctg taataaaccc gaaaattttg aatttttgta atttgttttt
     5341 gtaattcttt agtttgtatg tctgttgcta ttatgtctac tattctttcc cctgcactgt
     5401 accccccaat cccccctttt cttttaaaat tgtggatgaa tactgccatt tgtctgcaga
     5461 attggcgcac gcagtgccga tccgttcact aatcgaatgg atctgtctct gtctctctct
     5521 ccaccttctt cttctattcc ttcgggcctg tcgggtcccc tcggggttgg gaggtgggtc
     5581 tgaaacgata atggtgaata tccctgccta actctattca ctatagaaag tacagcaaaa
     5641 actattctta aacctaccaa gcctcctact atcattatga ataattttat ataccacagc
     5701 caatttgtta tgttaaacca attccacaaa cttgcccatt tatctaattc caataattct
     5761 tgttcattct tttcttgctg gttttgcgat tcttcaatta aggagtgtat taagcttgtg
     5821 taattgttaa tttctctgtc ccactccatc caggtcgtgt gattccaaat ctgttccaga
     5881 gatttattac tccaactagc attccaaggc acagcagtgg tgcaaatgag ttttccagag
     5941 caaccccaaa tccccaggag ctgttgatcc tttaggtatc tttccacagc caggattctt
     6001 gcctggagct gcttgatgcc ccagactgtg agttgcaaca gatgctgttg cgcctcaata
     6061 gccctcagca aattgttctg ctgctgcact ataccagaca ataattgtct ggcctgtacc
     6121 gtcagcgtca ttgacgctgc gcccatagtg cttcctgctg ctcccaagaa cccaaggaac
     6181 aaagctccta ttcccactgc tcttttttct ctctgcacca ctcttctctt tgccttggtg
     6241 ggtgctactc ctaatggttc aatttttact actttatatt tatataattc acttctccaa
     6301 ttgtccctca tatctcctcc tccaggtctg aagatcagcg gccggccgct tgctgtgcgg
     6361 tggtcttact tttgttttgc tcttcctcta tcttgtctaa agcttccttg gtgtctttta
     6421 tctctatcct ttgatgcaca caatagaggg ttgctactgt attatataat gatctaagtt
     6481 cttctgatcc tgtctgaagg gatggttgta gctgtcccag tatttgtcta cagccttctg
     6541 atgtttctaa caggccagga ttaactgcga atcgttctag ctccctgctt gcccatacta
     6601 tatgttttaa tttatatttt ttctttcccc ctggccttaa ccgaattttt tcccatcgcg
     6661 atctaattct cccccgctta atactgacgc tctcgcaccc atctctctcc ttctagcctc
     6721 cgctagtcaa aatttttggc gtactcacca gtcgccgccc ctcgcctctt gccgtgcgcg
     6781 cttcagcaag ccgagtcctg cgtcgagaga gctcctctgg tttccctttc gctttcaagt
     6841 ccctgttcgg gcgccactgc tagagatttt ccacactgac taaaagggtc tgagggatct
     6901 ctagttacca gagtcacaca acagacgggc acacactact tgaagcactc aaggcaagct
     6961 ttattgaggc ttaagcagtg ggttccctag ttagccagag agctcccagg ctcagatctg
     7021 gtctaaccag agagacccag tacaggcaaa acgcgctgct tatatagacc tcccaccgta
     7081 cacgcctacc gcccatttgc gtcaatgggg cggagttgtt acgacatttt ggaaagtccc
     7141 gttgattttg gtgccaaaac aaactcccat tgacgtcaat ggggtggaga cttggaaatc
     7201 cccgtgagtc aaaccgctat ccacgcccat tgatgtactg ccaaaaccgc atcaccatgg
     7261 taatagcgat gactaatacg tagatgtact gccaagtagg aaagtcccat aaggtcatgt
     7321 actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg ggcgtacttg
     7381 gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac tccacccatt
     7441 gacgtcaatg gaaagtccct attggcgtta ctatgggaac atacgtcatt attgacgtca
     7501 atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt tatgtaacgc
     7561 ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat taataactag
     7621 tcaataatca atgtcaacgc gtatatctgg cccgtacatc gcgaagcagc gcaaaacgcc
     7681 taaccctaag cagattcttc atgcaattgt cggtcaagcc ttgccttgtt gtagcttaaa
     7741 ttttgctcgc gcactactca gcgacctcca acacacaagc agggagcaga tactggctta
     7801 actatgcggc atcagagcag attgtactga gagtgcacca taggggatcg ggagatctcc
     7861 cgatccgtcg acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     7921 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     7981 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     8041 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     8101 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     8161 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     8221 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     8281 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     8341 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     8401 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     8461 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     8521 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     8581 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     8641 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     8701 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     8761 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     8821 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     8881 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     8941 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     9001 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     9061 aatctgctgc ttgcaaac
//
