LOCUS       sequence-128784-        7144 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constiutive mammalian expression of the bioluminescent GpNLuc 
            LumiFluor reporter for single-cell analysis and sensitive optical 
            imaging of living subjects..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7144)
  AUTHORS   Schaub FX, Reza MS, Flaveny CA, Li W, Musicant AM, Hoxha S, Guo M, 
            Cleveland JL, Amelio AL
  TITLE     Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical 
            Imaging and Flow Cytometry for Monitoring Tumorigenesis.
  JOURNAL   Cancer Res. 2015 Sep 30. pii: canres.3538.2014.
  PUBMED    26424696
REFERENCE   2  (bases 1 to 7144)
  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: "Cancer Res.
            2015 Sep 30. pii: canres.3538.2014."
COMMENT     Sequence Label: pRetroX-Tight-MCS_PGK-GpNLuc
FEATURES             Location/Qualifiers
     source          1..7144
                     /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"
     promoter        1604..1918
                     /label=tight TRE promoter
                     /note="Tet-responsive promoter PTight, consisting of seven 
                     tet operator sequences followed by the minimal CMV 
                     promoter"
     protein_bind    1612..1630
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1648..1666
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1683..1701
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1719..1737
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1755..1773
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1790..1808
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1826..1844
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1891..1915
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1974..2473
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2009..2032)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2406..2425
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             join(2488..2490,2491..2493,2494..3204)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2488..2490,2491..2493,2494..3204)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2533..2554)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2794..2813)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3141..3162
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3220..3735
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     LTR             3850..4275
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(4378..4400)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4536..4556)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4541..4870
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4721..4856
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4892..4911
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4992..5009)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5163..5751)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5243..5262)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5922..6713,6714..6782))
                     /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     6545..6564
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6783..6887)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6955..6973
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        join(7073..7144,1..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 ctgaggccct ttcgtcttca ctcgagttta ctccctatca
     1621 gtgatagaga acgtatgtcg agtttactcc ctatcagtga tagagaacga tgtcgagttt
     1681 actccctatc agtgatagag aacgtatgtc gagtttactc cctatcagtg atagagaacg
     1741 tatgtcgagt ttactcccta tcagtgatag agaacgtatg tcgagtttat ccctatcagt
     1801 gatagagaac gtatgtcgag tttactccct atcagtgata gagaacgtat gtcgaggtag
     1861 gcgtgtacgg tgggaggcct atataagcag agctcgttta gtgaaccgtc agatcgcctg
     1921 gagaaggatc cgcggccgcg ccggctctag atcgcgaacg cgtgaattct accgggtagg
     1981 ggaggcgctt ttcccaaggc agtctggagc atgcgcttta gcagccccgc tgggcacttg
     2041 gcgctacaca agtggcctct ggcctcgcac acattccaca tccaccggta ggcgccaacc
     2101 ggctccgttc tttggtggcc ccttcgcgcc accttctact cctcccctag tcaggaagtt
     2161 cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa atggaagtag cacgtctcac
     2221 tagtctcgtg cagatggaca gcaccgctga gcaatggaag cgggtaggcc tttggggcag
     2281 cggccaatag cagctttgct ccttcgcttt ctgggctcag aggctgggaa ggggtgggtc
     2341 cgggggcggg ctcaggggcg ggctcagggg cggggcgggc gcccgaaggt cctccggagg
     2401 cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg ctgttctcct cttcctcatc
     2461 tccgggcctt tcgacctgca gcccaagatg gtgagcaagg gcgaggagct gttcaccggg
     2521 gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc
     2581 ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc
     2641 ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc
     2701 ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa
     2761 ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc
     2821 gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc
     2881 aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc
     2941 tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac
     3001 atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac
     3061 ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac
     3121 cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact
     3181 ctcggcatgg acgagctgta caaggatatc tccggaggta tggtcttcac actcgaagat
     3241 ttcgttgggg actggcgaca gacagccggc tacaacctgg accaagtcct tgaacaggga
     3301 ggtgtgtcca gtttgtttca gaatctcggg gtgtccgtaa ctccgatcca aaggattgtc
     3361 ctgagcggtg aaaatgggct gaagatcgac atccatgtca tcatcccgta tgaaggtctg
     3421 agcggcgacc aaatgggcca gatcgaaaaa atttttaagg tggtgtaccc tgtggatgat
     3481 catcacttta aggtgatcct gcactatggc acactggtaa tcgacggggt tacgccgaac
     3541 atgatcgact atttcggacg gccgtatgaa ggcatcgccg tgttcgacgg caaaaagatc
     3601 actgtaacag ggaccctgtg gaacggcaac aaaattatcg acgagcgcct gatcaacccc
     3661 gacggctccc tgctgttccg agtaaccatc aacggagtga ccggctggcg gctgtgcgaa
     3721 cgcattctgg cgtaaatcga tagtggtcca ggctctagtt ttgactcaac aatatcacca
     3781 gctgaagcct atagagtacg agccatagat aaaataaaag attttattta gtctccagaa
     3841 aaagggggga atgaaagacc ccacctgtag gtttggcaag ctagcttaag taacgccatt
     3901 ttgcaaggca tggaaaaata cataactgag aatagagaag ttcagatcaa ggtcaggaac
     3961 agatggaaca gggtcgaccc tagagaacca tcagatgttt ccagggtgcc ccaaggacct
     4021 gaaatgaccc tgtgccttat ttgaactaac caatcagttc gcttctcgct tctgttcgcg
     4081 cgcttctgct ccccgagctc aataaaagag cccacaaccc ctcactcggg gcgccagtcc
     4141 tccgattgac tgagtcgccc gggtacccgt gtatccaata aaccctcttg cagttgcatc
     4201 cgacttgtgg tctcgctgtt ccttgggagg gtctcctctg agtgattgac tacccgtcag
     4261 cgggggtctt tcatttgggg gctcgtccgg gatcgggaga cccctgccca gggaccaccg
     4321 acccaccacc gggaggtaag ctggctgcct cgcgcgtttc ggtgatgacg gtgaaaacct
     4381 ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag
     4441 acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca
     4501 gtcacgtagc gatagcggag tgtagatccg gctgtggaat gtgtgtcagt tagggtgtgg
     4561 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4621 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4681 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     4741 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4801 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4861 cttttgcaaa aagcttactg gcttaactat gcggcatcag agcagattgt actgagagtg
     4921 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     4981 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     5041 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     5101 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     5161 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     5221 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     5281 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5341 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5401 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5461 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5521 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5581 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     5641 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5701 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5761 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5821 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5881 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5941 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     6001 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     6061 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     6121 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     6181 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca
     6241 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6301 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6361 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6421 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6481 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca
     6541 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6601 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6661 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6721 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6781 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6841 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6901 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     6961 cgtatcacga ggccctttcg tcttcaagaa ttagcttggc cattgcatac gttgtatcca
     7021 tatcataata tgtacattta tattggctca tgtccaacat taccgccatg ttgacattga
     7081 ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg
     7141 gagt
//
