LOCUS       sequence-211135-        6317 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  ChR2-YFP reporter vector of iTango and Cal-Light system for AAV 
            production.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6317)
  TITLE     fluorescent protein reporter vectors of iTango and Cal-Light system 
            for AAV production
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6317)
  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            "Unpublished"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: pAAV-TRE-FLEX-ChR2-YFP
FEATURES             Location/Qualifiers
     source          1..6317
                     /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
     protein_bind    join(298..316,317..339,340..358,359..381,382..400,401..423,
                     424..442,443..465,466..484,485..507,508..526,527..549,
                     550..568)
                     /label=tetracycline response element
                     /bound_moiety="tetracycline repressor TetR"
                     /note="contains seven copies of the tetracycline operator 
                     tetO"
     promoter        601..639
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    complement(778..811)
                     /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(862..895)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(931..1641,1642..1644))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(973..994)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1322..1341
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1581..1602
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(1657..2583)
                     /codon_start=1
                     /product="H134R mutant of an N-terminal fragment of 
                     channelrhodopsin-2 from the alga Chlamydomonas reinhardtii 
                     (Nagel et al., 2005)"
                     /label=ChR2(H134R)
                     /note="blue light-activated cation channel"
                     /translation="MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGT
                     NGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKN
                     PSMLYLATGHRVQWLRYAEWLLTCPVILIRLSNLTGLSNDYSRRTMGLLVSDIGTIVWG
                     ATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVS
                     WGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDI
                     RKTTKLNIGGTEIEVETLVEDEAEAGAV"
     protein_bind    2590..2623
                     /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    2674..2707
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2732..3320
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2785..2805)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3203..3214)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3355..3372)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3361..3568
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3591..3720
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     rep_origin      complement(3782..4370)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3862..3881)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4541..5332,5333..5401))
                     /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     5164..5183
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5402..5506)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5574..5592
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5630..5652)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5752..5771
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(5788..6243)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5925..5946)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6137..6156
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgaaacaa ttctgcagga attcggggcc
      181 gcggaggctg gatcggtccc ggtgtcttct atggaggtca aaacagcgtg gatggcgtct
      241 ccaggcgatc tgacggttca ctaaacgagc tctgcttata taggtcgagt ttaccactcc
      301 ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa
      361 agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagtttac
      421 cactccctat cagtgataga gaaaagtgaa agtcgagttt accactccct atcagtgata
      481 gagaaaagtg aaagtcgagt ttaccactcc ctatcagtga tagagaaaag tgaaagtcga
      541 gtttaccact ccctatcagt gatagagaaa agtgaaagtc gagctcggta cccgggtcga
      601 ggtaggcgtg tacggtggga ggcctatata agcagagctc gtttagtgaa ccgtcagatc
      661 gcctggagac gccatccacg ctgttttgac ctccatagaa gacaccggga ccgatccagc
      721 ctccgcggcc ccgaattcga gctcggtacc cggggatcct ctagagtcga ctccggaata
      781 acttcgtata ggatacttta tacgaagtta tgcagaatgg tagctggatt gtagctgcta
      841 ttagcaatat gaaacctctt aataacttcg tatagcatac attatacgaa gttatggcgc
      901 gcctcattac acctcgttct cgtagcagaa cttgtacagc tcgtccatgc cgagagtgat
      961 cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt tggggtcttt
     1021 gctcagggcg gactggtagc tcaggtagtg gttgtcgggc agcagcacgg ggccgtcgcc
     1081 gatgggggtg ttctgctggt agtggtcggc gagctgcacg ctgccgtcct cgatgttgtg
     1141 gcggatcttg aagttcacct tgatgccgtt cttctgcttg tcggccatga tatagacgtt
     1201 gtggctgttg tagttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc
     1261 gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg
     1321 ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt agccttcggg
     1381 catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggg cgaagcactg
     1441 caggccgtag ccgaaggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt
     1501 gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcgccct cgccggacac
     1561 gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt
     1621 gaacagctcc tcgcccttgc tcacggcggc cgctggcacg gctccggcct cggcttcgtc
     1681 ttcgacgaga gtctcgacct cgatctccgt tccgccgatg ttcagtttgg tggttttgcg
     1741 gatatctccg tgaatcaata tgtgctcgtg gatcaggacg cgcaggtagt gtcccaacaa
     1801 cccccaacaa tttttactca tcagatcaat aatcgtgtga cctacggtgg agccatagac
     1861 gctcaggacg ccaaaacctt cgggccccaa aatgaagaga attgggaaca taccccagct
     1921 cacgaaaaac agccatgcca tgccggtcac gacctggcgg caccgaccct ttggcacagt
     1981 atgataaccc tcgatatatg ctttggcggc gtgaaaaaat gtgttcgcgc catagcacaa
     2041 tccaagacaa aagaagatga ctttaacata gccggttgcc atggcgctgg tagcccccca
     2101 cacgatagtc ccgatgtctg agacaaggag tcccatggtt ctcctgctgt agtcgttgct
     2161 caggccggtg aggttgctca ggcggataag gatgacagga caagtgagca gccactctgc
     2221 atagcgcagc cactgcaccc ggtgtcctgt ggcaaggtag agcatagagg gattcttaaa
     2281 ctcaaaaaag aactcgagaa tcaccttaac catttcaatg gcgcacacat agatctcctc
     2341 ccagccgcat gtagatttcc aggtttggta ggcatagaac atcagcagca aaatgctgaa
     2401 tcctgctgca agccactgca ggacatttga cgcggtctga gcgccgttcg tgccgcgaga
     2461 ttcaatccat ccggcacagt aacattgatc ctcagggacc aggacggacc cgttcaccac
     2521 cacaggatta gtaacgaaca aaagttcgcg tccgacggca gacaaagcgc cgccatagtc
     2581 catgctagca taacttcgta taaagtatcc tatacgaagt tatttgcctt aacccagaaa
     2641 ttatcactgt tattctttag aatggtgcaa agaataactt cgtataatgt atgctatacg
     2701 aagttatgaa ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga
     2761 aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt
     2821 aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa
     2881 atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt
     2941 gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct
     3001 cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg
     3061 ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc
     3121 ggggaaatca tcgtcctttc cttggctgct cgcctatgtt gccacctgga ttctgcgcgg
     3181 gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct
     3241 gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc
     3301 cctttgggcc gcctccccgc atcgataccg agcgctgctc gagagatcga tctgcctcga
     3361 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     3421 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     3481 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     3541 gggaagacaa tagcaggcat gctggggaca cgtgcggacc gagcggccgc aggaacccct
     3601 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     3661 aaaggtcgcc cgacgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     3721 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3781 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3841 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3901 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3961 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4021 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4081 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4141 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4201 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4261 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     4321 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     4381 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4441 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4501 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4561 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4621 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4681 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4741 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4801 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4861 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4921 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4981 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5041 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5101 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5161 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5221 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     5281 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     5341 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     5401 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5461 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5521 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     5581 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     5641 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     5701 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     5761 agcagattgt actgagagtg caccataaaa ttgtaaacgt taatattttg ttaaaattcg
     5821 cgttaaattt ttgttaaatc agctcatttt ttaaccaata gaccgaaatc ggcaaaatcc
     5881 cttataaatc aaaagaatag cccgagatag agttgagtgt tgttccagtt tggaacaaga
     5941 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     6001 atggcccact acgtgaacca tcacccaaat caagtttttt ggggtcgagg tgccgtaaag
     6061 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     6121 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctaaggcg ctggcaagtg
     6181 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     6241 cgtactatgg ttgctttgac gtatgcggtg tgaaataccg cacagatgcg taaggagaaa
     6301 ataccgcatc aggcgcc
//
