LOCUS       sequence-79315-e        7175 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre dependent expression of oChIEF kinetic variant E163A/T199C with 
            physically uncoupled dTomato fluorophore .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7175)
  TITLE     ChIEF and oChIEF AAV vectors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7175)
  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            "Unpublished"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: 
            AAV-EF1a-DIO-oChIEF(E163A/T199C)-P2A-dTomato-WPRE-BGHpA
FEATURES             Location/Qualifiers
     source          1..7175
                     /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..1420
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1411
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1368..1388
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1451..1484)
                     /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(1535..1568)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1586..2287)
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(1669..1692)
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(1867..1886)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     2136..2154
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(2288..2344)
                     /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"
     regulatory      3433..3442
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3452..3485
                     /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    3536..3569
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3594..4182
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3647..3667)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4065..4076)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4215..4326
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4438..4578
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4438..4567
                     /label=AAV2 ITR
     rep_origin      4653..5108
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4740..4759)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4950..4971
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5125..5144)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5244..5266
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5304..5322)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5390..5494
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5495..5563,5564..6355)
                     /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(5713..5732)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6526..7114
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7015..7034
                     /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 tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg
      601 ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg
      661 gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc
      721 tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg
      781 gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc
      841 cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg
      901 agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc
      961 tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc
     1021 agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag
     1081 gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc
     1141 gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga
     1201 ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat
     1261 ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta
     1321 attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac
     1381 agtggttcaa agtttttttc ttccatttca ggtgtcgtga ggtaccggat cctctagagt
     1441 cgactccgga ataacttcgt ataggatact ttatacgaag ttatgcagaa tggtagctgg
     1501 attgtagctg ctattagcaa tatgaaacct cttaataact tcgtatagca tacattatac
     1561 gaagttatgg cgcgccgtcg acttacttgt acagctcgtc catgccgtac aggaacaggt
     1621 ggtggcggcc ctcggagcgc tcgtactgtt ccacgatggt gtagtcctcg ttgtgggagg
     1681 tgatgtccag cttggtgtcc acgtagtagt agccgggcag ttgcacgggc ttcttggcca
     1741 tgtagatggt cttgaactcc accaggtagt ggccgccgtc cttcagcttc agggcctggt
     1801 ggatctcgcc cttcagcacg ccgtcgcggg ggtacaggcg ctcggtggag gcctcccagc
     1861 ccatggtctt cttctgcatt acggggccgt cgggggggaa gttggtgccg cgcatcttca
     1921 ccttgtagat cagcgtgccg tcctgcaggg aggagtcctg ggtcacggtc accagaccgc
     1981 cgtcctcgaa gttcatcacg cgctcccact tgaagccctc ggggaaggac agcttcttgt
     2041 aatcggggat gtcggcgggg tgcttcacgt acgccttgga gccgtacatg aactgggggg
     2101 acaggatgtc ccaggcgaag ggcagggggc cgcccttggt caccttcagc ttggcggtct
     2161 gggtgccctc gtaggggcgg ccctcgccct cgccctcgat ctcgaactcg tggccgttca
     2221 tggagccctc catgcgcacc ttgaagcgca tgaactcttt gatgacctcc tcgcccttgc
     2281 tcaccattgg cccgggattc tcttcgacat cccctgcttg tttcaacagg gagaagttag
     2341 tggctccgct tccgttaacg ctgctctcgt acttgccggt gcccttgttc acggcgccgg
     2401 cctcggcctc gtcctccacc agggtctcca cctcgatctc ggtgccgccg atgttcagct
     2461 tggtggtctt tctgatgtcg ccgtggatca ggatgtgctc gtggatcagc actctcaggt
     2521 agtggcccag caggccccag cagttcttgc tcatcaggtc gatgatggtg tggcccacgg
     2581 tgctgccgta cacgctcagc acgccgaagc cctcggggcc caggatgaac aggatgggga
     2641 acatgcccca gctcacgaag aacagccagg ccatgccggt caccacctgt ctgcatctgc
     2701 ccttgggcac ggtgtggtag gcctcgatgt acaccttggc ggcgttgaag aaggtgtaga
     2761 tgccgtagca caggcccatc aggaagaaga tcactctcac gtagcccttg ctcagggcgg
     2821 cggtggtgcc ccacacgata cagccgatgt cgctcaccag caggcccatg gttctcttgt
     2881 tgtagtcgtt ggccaggccg gtcaggttgc tcaggtggat caggaccacg gggcaggtca
     2941 gcagccaggc ggcgtatctc agccacacgg tcttgttgcc gttgctgctg tagatcacgg
     3001 cgggctcgtc gaactcgtgg aagtactcta tgatgaactt gatcatctcg atggtggcca
     3061 cgtagatctc ctcccagccg caggtactct tccaggtctg gtagccgtag aacatcaggc
     3121 acagggcgct cagggcgaag gtgatccact gcaggatgtt ggcggccagc ttctcggcgt
     3181 tggtgccgtt actcttcagc caggccaggc agaagcactg gccgttgttg gggatgcaga
     3241 tcacgctgcc gttgttctcc agggtgtagc tggtctggaa cagcattctc tcgtgggctc
     3301 tgtggaacac gtagtcgggg ccgtcctggg tggccacggg cacggtggcg tcgctgccgg
     3361 tgctggcgcc ggcgctgccg gcggccaggg ccacggccag ggccagggcc agcagccagg
     3421 gtcttctgct caccatggtg gcgccgctag cataacttcg tataaagtat cctatacgaa
     3481 gttatttgcc ttaacccaga aattatcact gttattcttt agaatggtgc aaagaataac
     3541 ttcgtataat gtatgctata cgaagttatg aattcgatat caagcttatc gataatcaac
     3601 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta
     3661 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt
     3721 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg
     3781 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg
     3841 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca
     3901 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca
     3961 ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctatg
     4021 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag
     4081 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc
     4141 gccctcagac gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgagcgctgc
     4201 tcgagagatc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt
     4261 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat
     4321 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag
     4381 caagggggag gattgggaag acaatagcag gcatgcacgt gcggaccgag cggccgcagg
     4441 aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg
     4501 ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag
     4561 cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
     4621 tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc
     4681 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     4741 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     4801 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     4861 acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     4921 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     4981 caaccctatc tcgggctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     5041 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt
     5101 tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     5161 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     5221 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     5281 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     5341 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     5401 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     5461 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     5521 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     5581 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     5641 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     5701 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     5761 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     5821 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     5881 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     5941 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     6001 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     6061 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     6121 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     6181 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     6241 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     6301 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     6361 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     6421 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     6481 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     6541 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     6601 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     6661 accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     6721 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     6781 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     6841 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     6901 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     6961 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     7021 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     7081 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     7141 gttcctggcc ttttgctggc cttttgctca catgt
//
