LOCUS       sequence-163841-        7115 bp    DNA     circular SYN 14-APR-2025
DEFINITION  For AAV-TRE-Cre based Supernova-RFP, pK168 should be used with 
            pK170..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7115)
  AUTHORS   Luo W, Mizuno H, Iwata R, Nakazawa S, Yasuda K, Itohara S, Iwasato T
  TITLE     Supernova: A Versatile Vector System for Single-Cell Labeling and 
            Gene Function Studies in vivo.
  JOURNAL   Sci Rep. 2016 Oct 24;6:35747. doi: 10.1038/srep35747.
  PUBMED    27775045
REFERENCE   2  (bases 1 to 7115)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2016-10-24"; volume: "6:35747. doi"; pages: " 
            10.1038/srep35747"
COMMENT     Sequence Label: pK168.AAV-EF1-alpha-DIO-tTA-P2A-RFP-WPRE (Supernova)
FEATURES             Location/Qualifiers
     source          1..7115
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1450..1483)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(1534..1567)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1576..2271)
                     /codon_start=1
                     /product="red fluorescent protein from Entacmaea 
                     quadricolor"
                     /label=TurboRFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMKIKV
                     VEGGPLPFAFDILATSFMYGSKAFINHTQGIPDFFKQSFPEGFTWERITTYEDGGVLTA
                     TQDTSFQNGCIIYNVKINGVNFPSNGPVMQKKTRGWEANTEMLYPADGGLRGHSQMALK
                     LVGGGYLHCSFKTTYRSKKPAKNLKMPGFHFVDHRLERIKEADKETYVEQHEMAVAKYC
                     DLPSKLGHR"
     CDS             complement(2272..2328)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(join(2344..2349,2350..2385,2386..2388,2389..2424,
                     2425..2427,2428..2463,2464..2469,2470..3087))
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=tTA-Advanced
                     /note="In the Tet-Off(R) system, tTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the absence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     protein_bind    3100..3133
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    3184..3217
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3242..3830
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3295..3315)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3713..3724)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3862..4338
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4061..4080)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4378..4518
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4378..4507
                     /label=AAV2 ITR
     rep_origin      4593..5048
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4680..4699)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4890..4911
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5065..5084)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5184..5206
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5244..5262)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5330..5434
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5435..5503,5504..6295)
                     /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(5653..5672)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6466..7054
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6955..6974
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc
     1441 gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg
     1561 aagttatggc gcgcctcatc tgtgccccag tttgctaggg aggtcgcagt acttggccac
     1621 agccatctcg tgctgctcga cgtaggtctc tttgtcggcc tccttgattc tttccagtct
     1681 gtggtccacg aagtggaagc cgggcatctt gaggttctta gcgggtttct tggatctgta
     1741 tgtggtcttg aaggagcagt gcaggtagcc cccgcccacg agcttcaggg ccatctggct
     1801 gtggcctctc aggccgccgt cagcggggta cagcatctcg gtgttggcct cccagccgcg
     1861 tgttttcttc tgcatcacag ggccgttgga tgggaagttc accccgttga tcttgacgtt
     1921 gtagatgatg cagccgttct ggaagctggt gtcctgggta gcggtcagca cgcccccgtc
     1981 ttcgtatgtg gtgattctct cccatgtgaa gccctcaggg aaggactgct taaagaagtc
     2041 ggggatgccc tgggtgtggt tgatgaaggc tttgctgccg tacatgaagc tggtagccag
     2101 gatgtcgaag gcgaagggga gagggccgcc ctcgaccacc ttgatcttca tggtctgggt
     2161 gccctcgtag ggcttgcctt cgccctcgga tgtgcacttg aagtggtggt tgttcacggt
     2221 gccctccatg tacagcttca tgtgcatgtt ctccttgatc agctcgctca taggtccagg
     2281 gttctcctcc acgtctccag cctgcttcag caggctgaag ttagtagctc cgcttccgga
     2341 tcccccgggg agcatgtcaa ggtcaaaatc gtcaagagcg tcagcaggca gcatatcaag
     2401 gtcaaagtcg tcaagggcat cggctgggag catgtctaag tcaaaatcgt caagggcgtc
     2461 ggccggcccg ccgctttcgc actttagctg tttctccagg ccacatatga ttagttccag
     2521 gccgaaaagg aaggcaggtt cggctccctg atggtcgaac agctcaattg cttgtctcag
     2581 aagtgggggc atagaatcgg tggtaggtgt ctctctttcc tcttttgcta cttgatgctc
     2641 ctgatcctcc aatacgcagc ccagtgtaaa gtggcccacg gcggacagag cgtacagtgc
     2701 gttctccagg gagaagcctt gctgacacag gaacgcgagc tgattttcca gggtttcgta
     2761 ctgtttctct gttgggcggg tgccgagatg cactttagcc ccgtcgcgat gtgagaggag
     2821 agcacagcgg aatgacttgg cgttgttccg cagaaagtct tgccatgact cgccttccag
     2881 ggggcagaag tgggtatgat gcctgtccag catctcgatt gccagggcat cgagcagggc
     2941 ccgcttgttc ttcacgtgcc agtacagggt aggctgctca actcccagct tttgagcgag
     3001 tttccttgtc gtcaggcctt cgataccgac ttcattgagt aattccagag cagagtttat
     3061 gactttgctc ttgtccagtc tagacatggt ggcgctagca taacttcgta taaagtatcc
     3121 tatacgaagt tatttgcctt aacccagaaa ttatcactgt tattctttag aatggtgcaa
     3181 agaataactt cgtataatgt atgctatacg aagttatgaa ttcgatatca agcttatcga
     3241 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     3301 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     3361 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     3421 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     3481 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     3541 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     3601 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     3661 cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     3721 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     3781 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     3841 agcgctgctc gagagatcta cgggtggcat ccctgtgacc cctccccagt gcctctcctg
     3901 gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt aagttgcatc
     3961 attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt ggtatggagc
     4021 aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga accaagctgg
     4081 agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca agcgattctc
     4141 ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct cagctaattt
     4201 ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc caactcctaa
     4261 tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc gtgaaccact
     4321 gctcccttcc ctgtccttct gattttgtag gtaaccacgt gcggaccgag cggccgcagg
     4381 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     4441 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     4501 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4561 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     4621 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     4681 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4741 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4801 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4861 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4921 caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     4981 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     5041 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     5101 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5161 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5221 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     5281 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5341 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5401 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5461 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5521 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5581 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5641 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5701 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5761 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5821 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5881 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     5941 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6001 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6061 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6121 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6181 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6241 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6301 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6361 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6421 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6481 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6541 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6601 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6661 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6721 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6781 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6841 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6901 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     6961 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     7021 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     7081 gttcctggcc ttttgctggc cttttgctca catgt
//
