LOCUS       sequence-79317-e        7137 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre dependent expression of ChIEF kinetic variant E162A/T198C with 
            physically uncoupled dTomato fluorophore .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7137)
  TITLE     ChIEF and oChIEF AAV vectors
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7137)
  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            "Unpublished"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-ChIEF(E162A/T198C)-P2A-dTomato-WPRE-BGHpA
FEATURES             Location/Qualifiers
     source          1..7137
                     /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"
     protein_bind    3414..3447
                     /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    3498..3531
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3556..4144
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3609..3629)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4027..4038)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4177..4288
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   4400..4540
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4400..4529
                     /label=AAV2 ITR
     rep_origin      4615..5070
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4702..4721)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4912..4933
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5087..5106)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5206..5228
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5266..5284)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5352..5456
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5457..5525,5526..6317)
                     /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(5675..5694)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6488..7076
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6977..6996
                     /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 accgcgccag cctcggcctc gtcctccacc agcgtctcga
     2401 cctcaatctc agtgccacca atgttcaatt tggtggtctt gcgaatgtcg ccgtggatga
     2461 ggatatgctc gtggatcagc acgcgcaggt agtggccgag cagaccccag cagttcttcg
     2521 acatcaggtc aatgatggtg tggccgacgg tggagccgta cacgctcagg acgccgaagc
     2581 cctcggggcc gaggatgaac aggatgggga acatacccca tgatacgaag aagagccaag
     2641 ccatgccagt caccacctgg cgacaccggc ccttcggcac ggtgtggtac gcctcaatgt
     2701 agaccttggc tgcgttgaag aatgtgtaga tgccgtagca caggcccatc aggaagaaaa
     2761 tgacacggac gtatcccttg gacagcgcgg ccgtggtgcc ccacacgata cagccgatat
     2821 ctgacaccag cagacccatg gtacgcttgt tatagtcgtt cgccagaccc gtaaggttgc
     2881 tcagatggat aagaacgaca gggcaggtca gcagccaggc cgcgtaacga agccacacgg
     2941 tcttgttgcc gttggatgag tagatcaccg caggttcgtc aaactcatgg aaatactcga
     3001 tgatgaactt gatcatctcg atcgtggcca cgtaaatctc ctcccagccg caagtagact
     3061 tccaggtctg gtagccgtag aacatcaggc agagcgctga aagcgcaaaa gtaatccact
     3121 gcagaatgtt ggcagccaac ttctcggcat ttgttccgtt ggatttaagc caagccaagc
     3181 agaagcactg gccgttgttc gggatgcaaa taacagaacc attgttctca agagtgtatg
     3241 aggtttggaa gagcatgcgc tcgtgggcac ggtggaaaac gtagtcgggg ccatcctgag
     3301 tcgcgaccgg caccgtcgcg tcactgccag tcgaggctcc tgcgctgccg gccgccagcg
     3361 ccactgccag cgctagggca agaagccatg gcctccgcga catggtggct agcataactt
     3421 cgtataaagt atcctatacg aagttatttg ccttaaccca gaaattatca ctgttattct
     3481 ttagaatggt gcaaagaata acttcgtata atgtatgcta tacgaagtta tgaattcgat
     3541 atcaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     3601 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     3661 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct
     3721 ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct
     3781 gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc
     3841 gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg
     3901 acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc
     3961 tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac
     4021 gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg
     4081 cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc
     4141 ccgcatcgat accgagcgct gctcgagaga tctcgactgt gccttctagt tgccagccat
     4201 ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc
     4261 tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg
     4321 ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgcac
     4381 gtgcggaccg agcggccgca ggaaccccta gtgatggagt tggccactcc ctctctgcgc
     4441 gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg
     4501 gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg ggcgcctgat gcggtatttt
     4561 ctccttacgc atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc
     4621 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
     4681 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
     4741 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
     4801 tacggcacct cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc
     4861 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
     4921 tgttccaaac tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga
     4981 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     5041 attttaacaa aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg
     5101 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     5161 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     5221 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc
     5281 tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc
     5341 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5401 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5461 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5521 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5581 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5641 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5701 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     5761 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     5821 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     5881 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     5941 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6001 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6061 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6121 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6181 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6241 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6301 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6361 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6421 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6481 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6541 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6601 ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc
     6661 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6721 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     6781 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     6841 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     6901 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     6961 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     7021 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     7081 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgt
//
