LOCUS       pB-TopFlash-NLuc        9357 bp DNA     circular SYN 01-JUN-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9357)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 14, 2023 from SnapGene Viewer 7.0.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..9357
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1568..1599
                     /label=minP
                     /note="minimal TATA-box promoter with low basal activity"
     regulatory      1661..1670
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1667..2179
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             2183..2302
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     polyA_signal    2354..2475
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        2489..3651
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2720..3642
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     regulatory      3707..3716
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3713..4432
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     intron          4506..4735
                     /label=chimeric intron
                     /note="chimera between introns from adenovirus and 
                     immunoglobulin heavy chain genes"
     misc_feature    4793..5366
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             5399..5998
                     /codon_start=1
                     /gene="pac from Streptomyces"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVECPKDRATWCMTRKPGA"
     polyA_signal    6128..6262
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    6733..6766
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     primer_bind     complement(7136..7152)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    7160..7176
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(7184..7214)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7229..7250
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(7538..8126)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8297..9157)
                     /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"
     promoter        complement(9158..9262)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ctattattta accctagaaa
      421 gataatcata ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg catgtgtttt
      481 atcggtctgt atatcgaggt ttatttatta atttgaatag atattaagtt ttattatatt
      541 tacacttaca tactaataat aaattcaaca aacaatttat ttatgtttat ttatttatta
      601 aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aaacttttaa acattctctc
      661 ttttacaaaa ataaacttat tttgtacttt aaaaacagtc atgttgtaat cgataataag
      721 taattagctt aacctataca taatagaaac aaattatact tattagtcag tcagaaacaa
      781 ctttggcaca tatcaatatt atgctctcgt taattaactc gagggatccg gcgcgcctgt
      841 cattctaaat ctctctttca gcctaaagct ttttccccgt atccccccag gtgtctgcag
      901 gctcaaagag cagcgagaag cgttcagagg aaagcgatcc cgtgccacct tccccgtgcc
      961 cgggctgtcc ccgcacgctg ccggctcggg gatgcggggg gagcgccgga ccggagcgga
     1021 gccccgggcg gctcgctgct gccccctagc gggggaggga cgtaattaca tccctggggg
     1081 ctttgggagg gggctgtccc cgtgagctcc caggcgcgcc tgtcattcta aatctctctt
     1141 tcagcctaaa gctttttccc cgtatccccc caggtgtctg caggctcaaa gagcagcgag
     1201 aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg tccccgcacg
     1261 ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg gcggctcgct
     1321 gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg ggggggctgt
     1381 ccccgtgagc tcccaggcgc gccttaaggt accgagctct tacgcgagat caaagggggt
     1441 aagatcaaag ggggtaagat caaaggggcg cgagatcaaa gggggtaaga tcaaaggggg
     1501 taagatcaaa gggggtaaga tcaaaggggc gcgcccgcgt gctagcccgg gctcgagatc
     1561 tagactctag agggtatata atggaagctc gaattccagc ttggcattcc ggtactgttg
     1621 gtaaaaagct tacgcgtggc aatccggtac tgttggtaaa gccaccatgg tcttcacact
     1681 cgaagatttc gttggggact ggcgacagac agccggctac aacctggacc aagtccttga
     1741 acagggaggt gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc cgatccaaag
     1801 gattgtcctg agcggtgaaa atgggctgaa gatcgacatc catgtcatca tcccgtatga
     1861 aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg tgtaccctgt
     1921 ggatgatcat cactttaagg tgatcctgca ctatggcaca ctggtaatcg acggggttac
     1981 gccgaacatg atcgactatt tcggacggcc gtatgaaggc atcgccgtgt tcgacggcaa
     2041 aaagatcact gtaacaggga ccctgtggaa cggcaacaaa attatcgacg agcgcctgat
     2101 caaccccgac ggctccctgc tgttccgagt aaccatcaac ggagtgaccg gctggcggct
     2161 gtgcgaacgc attctggcga attctcacgg ctttccgcct gaggttgaag agcaagccgc
     2221 cggtacattg cctatgtcct gcgcacaaga aagcggtatg gaccggcacc cagccgcttg
     2281 tgcttcagct cgcatcaacg tctaaggccg cgactctaga gtcggggcgg ccggccgctt
     2341 cgagcagaca tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga
     2401 aaaaaatgct ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc
     2461 tgcaataaac aagttgctag cttcgtgagg ctccggtgcc cgtcagtggg cagagcgcac
     2521 atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgaaccg gtgcctagag
     2581 aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc tttttcccga
     2641 gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt ttcgcaacgg
     2701 gtttgccgcc agaacacagg taagtgccgt gtgtggttcc cgcgggcctg gcctctttac
     2761 gggttatggc ccttgcgtgc cttgaattac ttccacctgg ctccagtacg tgattcttga
     2821 tcccgagctg gagccagggg cgggccttgc gctttaggag ccccttcgcc tcgtgcttga
     2881 gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc
     2941 tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc tgctgcgacg
     3001 ctttttttct ggcaagatag tcttgtaaat gcgggccagg atctgcacac tggtatttcg
     3061 gtttttgggc ccgcggccgg cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg
     3121 cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc tggccggcct
     3181 gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc aaggctggcc
     3241 cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc tccagggggc
     3301 tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac acaaaggaaa
     3361 agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc
     3421 aggcacctcg attagttctg gagcttttgg agtacgtcgt ctttaggttg gggggagggg
     3481 ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag gccagcttgg
     3541 cacttgatgt aattctcgtt ggaatttgcc ctttttgagt ttggatcttg gttcattctc
     3601 aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg aacacgtggt
     3661 cgcggccaat tctgcagtcg agctcaagct tcgaattcca ccggtcgcca ccatggtgag
     3721 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     3781 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     3841 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     3901 caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga
     3961 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     4021 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     4081 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     4141 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     4201 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     4261 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     4321 cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     4381 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt aaaggatccc
     4441 tcgaggcggc cgcgggcgcc agtgtgctgg aattaattcg ctgtctgcga gggccagctg
     4501 ttggggtgag tactccctct caaaagcggg catgacttct gcgctaagat tgtcagtttc
     4561 caaaaacgag gaggatttga tattcacctg gcccgcggtg atgcctttga gggtggccgc
     4621 gtccatctgg tcagaaaaga caatcttttt gttgtcaagc ttgaggtgtg gcaggcttga
     4681 gatctggcca tacacttgag tgacaatgac atccactttg cctttctctc cacaggtgtc
     4741 cactcccagg tccaactgca ggtcgagcat gcatctaggg cggccaattc cgcccctctc
     4801 cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg tgtgcgtttg
     4861 tctatatgtg attttccacc atattgccgt cttttggcaa tgtgagggcc cggaaacctg
     4921 gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa ggaatgcaag
     4981 gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga caaacaacgt
     5041 ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc ctctgcggcc
     5101 aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc cacgttgtga
     5161 gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac aaggggctga
     5221 aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg tgcacatgct
     5281 ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac ggggacgtgg
     5341 ttttcctttg aaaaacacga tgataagctt gccacaaccc acaaggagac gaccttccat
     5401 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc gggccgtacg
     5461 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg acccggaccg
     5521 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     5581 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     5641 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     5701 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     5761 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     5821 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     5881 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     5941 cgtcgagtgc ccgaaggacc gcgcgacctg gtgcatgacc cgcaagcccg gtgcctgacg
     6001 cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg gctccgaccg
     6061 aagccgaccc gggcggcccc gccgaccccg cacccgcccc cgaggcccac cgactctaga
     6121 gctcgctgat ccagacatga taagatacat tgatgagttt ggacaaacca caactagaat
     6181 gcagtgaaaa aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat
     6241 tataagctgc aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca
     6301 gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtatggctga
     6361 ttatgatccg gctgcctcgc gcttaattaa cccgggggcc gcctgggaac tgccgctggc
     6421 cccccaccgc cccaaggatc tccggtcccc gcccggcttg ctgacgtcac ggatgcccca
     6481 gggtgtgctg ggcaggtagc gggagcctgg gaaatggagt ccattagcag aagtggccac
     6541 ttccaggagt cgctgtgccc cgatgcacac tgggaagtcc gcagctccga ggcccccagt
     6601 ggaaatcgcc agatgagggc ctcctccggg gaatgctggg aaatggagtc tacaggccgg
     6661 aggggtcccc acggcatact agcaagtgtg tgtagcaccg cgtaaagggg atgaatagca
     6721 gactgccccg ggataacttc gtataggata ctttatacga agttatgagc tctgtgatta
     6781 ggaattgata ctagtcggtc acgatatcta taacaagaaa atatatatat aataagttat
     6841 cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc ttaaaagtca
     6901 cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca aaatcagtga
     6961 cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg agatgtccta
     7021 aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca agaatgcatg
     7081 cgtcaatttt acgcagacta tctttctagg gttaaaaaag cttggcgtaa tcatggtcat
     7141 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     7201 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     7261 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     7321 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     7381 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     7441 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     7501 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     7561 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     7621 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     7681 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     7741 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     7801 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     7861 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     7921 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     7981 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     8041 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     8101 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     8161 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     8221 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     8281 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     8341 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     8401 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     8461 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     8521 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     8581 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     8641 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     8701 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     8761 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     8821 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     8881 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     8941 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     9001 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     9061 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     9121 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     9181 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     9241 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa
     9301 ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtc
//
