LOCUS       Exported                7135 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Constiutive mammalian expression of the bioluminescent OgNLuc 
            LumiFluor reporter for single-cell analysis and sensitive optical 
            imaging of living subjects..
ACCESSION   .
VERSION     .
KEYWORDS    pRetroX-Tight-MCS_PGK-OgNLuc
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7135)
  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 7135)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7135
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        310..513
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     464..484
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     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             2488..3195
                     /codon_start=1
                     /product="monomeric orange fluorescent protein with a large
                     Stokes shift (Shcherbakova et al., 2012)"
                     /label=LSSmOrange
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEENNMAIIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEG
                     FQTVKLKVTKGGPLPFAWDILSPQFTYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGMEASSERMYPEDGALK
                     GEDKLRLKLKDGGHYTSEVKTTYKAKKPVQLPGAYIVDIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     2901..2920
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             3211..3726
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     LTR             3841..4266
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(4369..4391)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4527..4547)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4532..4861
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4712..4847
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4774..4793
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     4883..4902
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4983..5000)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5154..5742)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5234..5253)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5913..6773)
                     /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     6536..6555
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6774..6878)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6946..6964
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        7064..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 gcgaggagaa taacatggcc
     2521 atcatcaagg agttcatgcg cttcaaggtg cgcatggagg gctccgtgaa cggccacgag
     2581 ttcgagatcg agggcgaggg cgagggccgc ccctacgagg gctttcagac cgttaagctg
     2641 aaggtgacca agggtggccc cctgcccttc gcctgggaca tcttgtcccc tcagttcacc
     2701 tacggctcca aggcctacgt gaagcacccc gccgacatcc ccgactacct caagctgtcc
     2761 ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc
     2821 gtgactcagg actcctccct gcaggacggc gagttcatct acaaggtgaa gctgcgcggc
     2881 accaacttcc cctccgacgg ccccgtaatg cagaagaaga ccatgggcat ggaggcctcc
     2941 tccgagcgga tgtaccccga ggacggcgcc ctgaagggcg aggacaagct caggctgaag
     3001 ctgaaggacg gcggccacta cacctccgag gtcaagacca cctacaaggc caagaagccc
     3061 gtgcagttgc ccggcgccta catcgtcgac atcaagttgg acatcacctc ccacaacgag
     3121 gactacacca tcgtggaaca gtacgaacgc gccgagggcc gccactccac cggcggcatg
     3181 gacgagctgt acaaggatat ctccggaggt atggtcttca cactcgaaga tttcgttggg
     3241 gactggcgac agacagccgg ctacaacctg gaccaagtcc ttgaacaggg aggtgtgtcc
     3301 agtttgtttc agaatctcgg ggtgtccgta actccgatcc aaaggattgt cctgagcggt
     3361 gaaaatgggc tgaagatcga catccatgtc atcatcccgt atgaaggtct gagcggcgac
     3421 caaatgggcc agatcgaaaa aatttttaag gtggtgtacc ctgtggatga tcatcacttt
     3481 aaggtgatcc tgcactatgg cacactggta atcgacgggg ttacgccgaa catgatcgac
     3541 tatttcggac ggccgtatga aggcatcgcc gtgttcgacg gcaaaaagat cactgtaaca
     3601 gggaccctgt ggaacggcaa caaaattatc gacgagcgcc tgatcaaccc cgacggctcc
     3661 ctgctgttcc gagtaaccat caacggagtg accggctggc ggctgtgcga acgcattctg
     3721 gcgtaaatcg atagtggtcc aggctctagt tttgactcaa caatatcacc agctgaagcc
     3781 tatagagtac gagccataga taaaataaaa gattttattt agtctccaga aaaagggggg
     3841 aatgaaagac cccacctgta ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc
     3901 atggaaaaat acataactga gaatagagaa gttcagatca aggtcaggaa cagatggaac
     3961 agggtcgacc ctagagaacc atcagatgtt tccagggtgc cccaaggacc tgaaatgacc
     4021 ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc gcgcttctgc
     4081 tccccgagct caataaaaga gcccacaacc cctcactcgg ggcgccagtc ctccgattga
     4141 ctgagtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat ccgacttgtg
     4201 gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca gcgggggtct
     4261 ttcatttggg ggctcgtccg ggatcgggag acccctgccc agggaccacc gacccaccac
     4321 cgggaggtaa gctggctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     4381 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     4441 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     4501 cgatagcgga gtgtagatcc ggctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     4561 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     4621 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     4681 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     4741 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     4801 ggcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     4861 aaagcttact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg
     4921 cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
     4981 tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
     5041 tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga
     5101 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat
     5161 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     5221 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     5281 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     5341 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     5401 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     5461 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     5521 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     5581 ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     5641 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     5701 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
     5761 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga
     5821 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc
     5881 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct
     5941 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata
     6001 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca
     6061 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga
     6121 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga
     6181 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctgc aggcatcgtg
     6241 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga
     6301 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt
     6361 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct
     6421 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca
     6481 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaac acgggataat
     6541 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6601 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6661 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6721 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6781 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     6841 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     6901 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     6961 aggccctttc gtcttcaaga attagcttgg ccattgcata cgttgtatcc atatcataat
     7021 atgtacattt atattggctc atgtccaaca ttaccgccat gttgacattg attattgact
     7081 agttattaat agtaatcaat tacggggtca ttagttcata gcccatatat ggagt
//
