LOCUS       sequence-207774-        8696 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gamma-Retroviral transfer vector for expressing HuR (wild type), V5 
            and mKO2 tags, IRES-driven Puromycin selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8696)
  AUTHORS   Sandra Martha Gomes Dias
  TITLE     HuR (ELAVL1) silencing sensitizes triple-negative breast cancer to 
            glutaminase-targeted therapy
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8696)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pQC V5 mKO2 HuR.wt IRES Puro
FEATURES             Location/Qualifiers
     source          1..8696
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        310..513
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             514..689
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    752..951
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1152..1568
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1498..1520
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1536..1552
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     enhancer        1605..1908
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1909..2112
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2109..2133
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2267..2308
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2318..2971
                     /codon_start=1
                     /product="monomeric Kusabira-Orange 2 fluorescent protein"
                     /label=mKO2
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     CDS             2318..2971
                     /codon_start=1
                     /product="humanized monomeric Kusabira-Orange fluorescent 
                     protein"
                     /label=hmKO
                     /note="mammalian codon-optimized"
                     /translation="MVSVIKPEMKMRYYMDGSVNGHEFTIEGEGTGRPYEGHQEMTLRV
                     TMAEGGPMPFAFDLVSHVFCYGHRVFTKYPEEIPDYFKQAFPEGLSWERSLEFEDGGSA
                     SVSAHISLRGNTFYHKSKFTGVNFPADGPIMQNQSVDWEPSTEKITASDGVLKGDVTMY
                     LKLEGGGNHKCQMKTTYKAAKEILEMPGDHYIGHRLVRKTEGNITEQVEDAVAHS"
     misc_feature    4025..4597
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4192..4209)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4419..4438
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4634..5233
                     /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(4634..4653)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5130..5150
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5402..5827
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(5930..5952)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6088..6108)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        6093..6422
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6273..6408
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6444..6463
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(6544..6561)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6715..7303)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6795..6814)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7474..8265,8266..8334))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8097..8116
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8335..8439)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     8507..8525
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(8625..8696,1..309)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
       61 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      121 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      181 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgctctg gcattatgcc
      241 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      301 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      361 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      421 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      481 cgtgtacggt gggaggtcta tataagcaga gctcaataaa agagcccaca acccctcact
      541 cggcgcgcca gtcttccgat agactgcgtc gcccgggtac ccgtattccc aataaagcct
      601 cttgctgttt gcatccgaat cgtggtctcg ctgttccttg ggagggtctc ctctgagtga
      661 ttgactaccc acgacggggg tctttcattt gggggctcgt ccgggatttg gagacccctg
      721 cccagggacc accgacccac caccgggagg taagctggcc agcaacttat ctgtgtctgt
      781 ccgattgtct agtgtctatg tttgatgtta tgcgcctgcg tctgtactag ttagctaact
      841 agctctgtat ctggcggacc cgtggtggaa ctgacgagtt ctgaacaccc ggccgcaacc
      901 ctgggagacg tcccagggac tttgggggcc gtttttgtgg cccgacctga ggaagggagt
      961 cgatgtggaa tccgaccccg tcaggatatg tggttctggt aggagacgag aacctaaaac
     1021 agttcccgcc tccgtctgaa tttttgcttt cggtttggaa ccgaagccgc gcgtcttgtc
     1081 tgctgcagcg ctgcagcatc gttctgtgtt gtctctgtct gactgtgttt ctgtatttgt
     1141 ctgaaaatta gggccagact gttaccactc ccttaagttt gaccttaggt cactggaaag
     1201 atgtcgagcg gatcgctcac aaccagtcgg tagatgtcaa gaagagacgt tgggttacct
     1261 tctgctctgc agaatggcca acctttaacg tcggatggcc gcgagacggc acctttaacc
     1321 gagacctcat cacccaggtt aagatcaagg tcttttcacc tggcccgcat ggacacccag
     1381 accaggtccc ctacatcgtg acctgggaag ccttggcttt tgacccccct ccctgggtca
     1441 agccctttgt acaccctaag cctccgcctc ctcttcctcc atccgccccg tctctccccc
     1501 ttgaacctcc tcgttcgacc ccgcctcgat cctcccttta tccagccctc actccttctc
     1561 taggcgccgg aattgaagat ctgggggatc gatcctctag agtccgttac ataacttacg
     1621 gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg
     1681 tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta
     1741 cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt
     1801 gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac
     1861 tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt
     1921 tggcagtaca tcaatgggcg tgaatagcgg tttgactcac ggggatttcc aagtctccac
     1981 cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt
     2041 cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat
     2101 ataagcagag ctcgtttagt gaaccgtcag atcgcctgga gacgccatcc acgctgtttt
     2161 gacctccata gaagacaccg ggaccgatcc agcctccgcg gccgggaacg gtgcattgga
     2221 acggacctgc aggaattgat ccgcggccgc accggggatc accatgggta agcctatccc
     2281 taaccctctc ctcggtctcg attctacggg atctaccatg gtgagcgtga tcaagcccga
     2341 gatgaagatg aggtactaca tggacggctc cgtcaatggg catgagttca caatcgaggg
     2401 tgagggcaca ggcagacctt acgagggaca tcaggagatg acactgcgcg tcacaatggc
     2461 cgagggcggg ccaatgcctt tcgccttcga cctggtgtcc cacgtgttct gttacggcca
     2521 cagagttttt accaagtacc cagaagagat cccagactat ttcaagcagg cctttcctga
     2581 gggcctgtcc tgggagaggt ccctggagtt cgaggacggc ggctccgcct ccgtgagcgc
     2641 ccacatcagc ctgaggggca acaccttcta ccacaagtcc aagttcaccg gcgtgaactt
     2701 ccccgccgac ggccccatca tgcagaacca gagcgtggac tgggagccct ccaccgagaa
     2761 gatcaccgcc agcgacggcg tgctgaaggg cgacgtgacc atgtacctga agctggaggg
     2821 cggcggcaac cacaagtgcc agatgaagac cacctacaag gccgccaagg agatcctgga
     2881 gatgcccggc gaccactaca tcggccacag gctggtgagg aagaccgagg gcaacatcac
     2941 cgagcaggtg gaggacgccg tggcccactc cggtggcgga ggctcgggcg gaggtgggtc
     3001 gggatccggt tctagagcca ccggtatgtc taatggttat gaagaccaca tggccgaaga
     3061 ctgcaggggt gacatcggga gaacgaattt gatcgtcaac tacctccctc agaacatgac
     3121 ccaggatgag ttacgaagcc tgttcagcag cattggtgaa gttgaatctg caaaacttat
     3181 tcgggataaa gtagcaggac acagcttggg ctatggcttt gtgaactacg tgaccgcgaa
     3241 ggatgcagag agagcgatca acacgctgaa cggcttgagg ctccagtcaa aaaccattaa
     3301 ggtgtcgtat gctcgcccga gctcagaggt gatcaaagac gccaacttgt acatcagcgg
     3361 gctcccgcgg accatgaccc agaaggacgt agaagacatg ttctctcggt ttgggcggat
     3421 catcaactcg cgggtcctcg tggatcagac tacaggtttg tccagagggg ttgcgtttat
     3481 ccggtttgac aaacggtcgg aggcagaaga ggcaattacc agtttcaatg gtcataaacc
     3541 cccaggttcc tctgagccca tcacagtgaa gtttgcagcc aaccccaacc agaacaaaaa
     3601 cgtggcactc ctctcgcagc tgtaccactc gccagcgcga cggttcggag gccccgttca
     3661 ccaccaggcg cagagattca ggttctcccc catgggcgtc gatcacatga gcgggctctc
     3721 tggcgtcaac gtgccaggaa acgcctcctc cggctggtgc attttcatct acaacctggg
     3781 gcaggatgcc gacgagggga tcctctggca gatgtttggg ccgtttggtg ccgtcaccaa
     3841 tgtgaaagtg atccgcgact tcaacaccaa caagtgcaaa gggtttggct ttgtgaccat
     3901 gacaaactat gaagaagccg cgatggccat agccagcctg aacggctacc gcctggggga
     3961 caaaatctta caggtttcct tcaaaaccaa caagtcccac aaataagaat tctgattaat
     4021 tccgcccctc tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc
     4081 ggtgtgcgtt tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg
     4141 cccggaaacc tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca
     4201 aaggaatgca aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa
     4261 gacaaacaac gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt
     4321 gcctctgcgg ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt
     4381 gccacgttgt gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca
     4441 acaaggggct gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc
     4501 ggtgcacatg ctttacatgt gtttagtcga ggttaaaaaa cgtctaggcc ccccgaacca
     4561 cggggacgtg gttttccttt gaaaaacacg atgataagct tgatcacgcg tctcgagatc
     4621 accggagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt
     4681 ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac
     4741 cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg
     4801 cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg
     4861 gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc
     4921 cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca
     4981 ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg
     5041 caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt
     5101 gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt
     5161 caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa
     5221 gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga gcgcacgacc
     5281 ccatgcatcg atcagtggtc caggctctag ttttgactca acaatatcac cagctgaagc
     5341 ctatagagta cgagccatag ataaaataaa agattttatt tagtctccag aaaaaggggg
     5401 gaatgaaaga ccccacctgt aggtttggca agctagctta agtaacgcca ttttgcaagg
     5461 catggaaaaa tacataactg agaatagaga agttcagatc aaggtcagga acagatggaa
     5521 cagggtcgac cctagagaac catcagatgt ttccagggtg ccccaaggac ctgaaatgac
     5581 cctgtgcctt atttgaacta accaatcagt tcgcttctcg cttctgttcg cgcgcttctg
     5641 ctccccgagc tcaataaaag agcccacaac ccctcactcg gggcgccagt cctccgattg
     5701 actgagtcgc ccgggtaccc gtgtatccaa taaaccctct tgcagttgca tccgacttgt
     5761 ggtctcgctg ttccttggga gggtctcctc tgagtgattg actacccgtc agcgggggtc
     5821 tttcatttgg gggctcgtcc gggatcggga gacccctgcc cagggaccac cgacccacca
     5881 ccgggaggta agctggctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     5941 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     6001 gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta
     6061 gcgatagcgg agtgtagatc cggctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     6121 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     6181 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     6241 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     6301 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     6361 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     6421 aaaagcttac tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccatat
     6481 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gctcttccgc
     6541 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     6601 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     6661 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     6721 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     6781 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6841 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6901 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6961 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     7021 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     7081 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     7141 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     7201 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     7261 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     7321 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     7381 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     7441 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     7501 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     7561 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     7621 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     7681 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     7741 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgctg caggcatcgt
     7801 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7861 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7921 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7981 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     8041 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa cacgggataa
     8101 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     8161 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     8221 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     8281 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     8341 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     8401 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     8461 acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac
     8521 gaggcccttt cgtcttcaag aattagcttg gccattgcat acgttgtatc catatcataa
     8581 tatgtacatt tatattggct catgtccaac attaccgcca tgttgacatt gattattgac
     8641 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagt
//
