LOCUS       sequence-412234-        5528 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses the membrane-localized enhanced green fluorescent protein 
            EGFP-CAAX in a cre-dependent manner.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5528)
  AUTHORS   Wang H, Qian T, Zhao Y, Zhuo Y, Wu C, Osakada T, Chen P, Chen Z, Ren
            H, Yan Y, Geng L, Fu S, Mei L, Li G, Wu L, Jiang Y, Qian W, Zhang L,
            Peng W, Xu M, Hu J, Jiang M, Chen L, Tang C, Zhu Y, Lin D, Zhou JN, 
            Li Y
  TITLE     A tool kit of highly selective and sensitive genetically encoded 
            neuropeptide sensors.
  JOURNAL   Science. 2023 Nov 17;382(6672):eabq8173. doi: 
            10.1126/science.abq8173. Epub 2023 Nov 17.
  PUBMED    37972184
REFERENCE   2  (bases 1 to 5528)
  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: "Science."; 
            date: "2023-11-17"; volume: "382(6672):eabq8173. doi"; pages: " 
            10.1126/science.abq8173. Epub 2023 Nov 17"
COMMENT     Sequence Label: pAAV-hSyn-DIO-EGFP-CAAX
FEATURES             Location/Qualifiers
     source          1..5528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     66..85
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   227..356
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    complement(831..864)
                     /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(915..948)
                     /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(1010..1720,1721..1723,1724..1726))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1010..1720,1721..1723,1724..1726))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1052..1073)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1401..1420
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1660..1681
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1723..1732
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1739..1772
                     /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    1823..1856
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1881..2469
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(1934..1954)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2352..2363)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    2501..2977
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(2700..2719)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3017..3157
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3017..3146
                     /label=AAV2 ITR
     rep_origin      3232..3687
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3319..3338)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3529..3550
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3704..3723)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3823..3845
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3883..3901)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3969..4073
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4074..4142,4143..4934)
                     /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(4292..4311)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5105..5528,1..165)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
       61 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
      121 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
      181 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgtcctg caggcagctg
      241 cgcgctcgct cgctcactga ggccgcccgg gcgtcgggcg acctttggtc gcccggcctc
      301 agtgagcgag cgagcgcgca gagagggagt ggccaactcc atcactaggg gttcctgcgg
      361 ccgcacgcgt atgaggcggg gtgggggtgc ctacctgacg accgaccccg acccactgga
      421 caagcaccca acccccattc cccaaattgc gcatccccta tcagagaggg ggaggggaaa
      481 caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag tgccttcgcc
      541 cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga cgtcactcgc
      601 cggtcccccg caaactcccc ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc
      661 agccggaccg caccacgcga ggcgcgagat aggggggcac gggcgcgacc atctgcgctg
      721 cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg
      781 cctgagagcg cagtcgagaa accggtgcca cctctagagt cgactccgga ataacttcgt
      841 ataggatact ttatacgaag ttatgcagaa tggtagctgg attgtagctg ctattagcaa
      901 tatgaaacct cttaataact tcgtatagca tacattatac gaagttatgg cgcatatgtt
      961 acataattac acactttgtc tttgacttct ttttcttctt tttaccatcc ttgtacagct
     1021 cgtccatgcc gagagtgatc ccggcggcgg tcacgaactc cagcaggacc atgtgatcgc
     1081 gcttctcgtt ggggtctttg ctcagggcgg actgggtgct caggtagtgg ttgtcgggca
     1141 gcagcacggg gccgtcgccg atgggggtgt tctgctggta gtggtcggcg agctgcacgc
     1201 tgccgtcctc gatgttgtgg cggatcttga agttcacctt gatgccgttc ttctgcttgt
     1261 cggccatgat atagacgttg tggctgttgt agttgtactc cagcttgtgc cccaggatgt
     1321 tgccgtcctc cttgaagtcg atgcccttca gctcgatgcg gttcaccagg gtgtcgccct
     1381 cgaacttcac ctcggcgcgg gtcttgtagt tgccgtcgtc cttgaagaag atggtgcgct
     1441 cctggacgta gccttcgggc atggcggact tgaagaagtc gtgctgcttc atgtggtcgg
     1501 ggtagcggct gaagcactgc acgccgtagg tcagggtggt cacgagggtg ggccagggca
     1561 cgggcagctt gccggtggtg cagatgaact tcagggtcag cttgccgtag gtggcatcgc
     1621 cctcgccctc gccggacacg ctgaacttgt ggccgtttac gtcgccgtcc agctcgacca
     1681 ggatgggcac caccccggtg aacagctcct cgcccttgct caccatggtg gcgctagcat
     1741 aacttcgtat aaagtatcct atacgaagtt atttgcctta acccagaaat tatcactgtt
     1801 attctttaga atggtgcaaa gaataacttc gtataatgta tgctatacga agttatgaat
     1861 tcgatatcaa gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg
     1921 gtattcttaa ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt
     1981 atcatgctat tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc
     2041 tgtctcttta tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
     2101 ttgctgacgc aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga
     2161 ctttcgcttt ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct
     2221 gctggacagg ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat
     2281 cgtcctttcc ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct
     2341 gctacgtccc ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc
     2401 tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg
     2461 cctccccgca tcgataccga gcgctgctcg agagatctac gggtggcatc cctgtgaccc
     2521 ctccccagtg cctctcctgg ccctggaagt tgccactcca gtgcccacca gccttgtcct
     2581 aataaaatta agttgcatca ttttgtctga ctaggtgtcc ttctataata ttatggggtg
     2641 gaggggggtg gtatggagca aggggcaagt tgggaagaca acctgtaggg cctgcggggt
     2701 ctattgggaa ccaagctgga gtgcagtggc acaatcttgg ctcactgcaa tctccgcctc
     2761 ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg ttgggattcc aggcatgcat
     2821 gaccaggctc agctaatttt tgtttttttg gtagagacgg ggtttcacca tattggccag
     2881 gctggtctcc aactcctaat ctcaggtgat ctacccacct tggcctccca aattgctggg
     2941 attacaggcg tgaaccactg ctcccttccc tgtccttctg attttgtagg taaccacgtg
     3001 cggaccgagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     3061 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     3121 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     3181 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     3241 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3301 ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3361 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3421 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     3481 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3541 tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta taagggattt
     3601 tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3661 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     3721 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3781 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     3841 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     3901 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     3961 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4021 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4081 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4141 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4201 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4261 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4321 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4381 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4441 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     4501 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     4561 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     4621 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     4681 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     4741 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     4801 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     4861 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     4921 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     4981 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     5041 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     5101 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     5161 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     5221 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     5281 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     5341 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     5401 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     5461 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     5521 aagggaga
//
