LOCUS       sequence-367118-        6311 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Double floxed EGFP under the control of EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6311)
  AUTHORS   Xu J, Jo A, DeVries RP, Deniz S, Cherian S, Sunmola I, Song X, 
            Marshall JJ, Gruner KA, Daigle TL, Contractor A, Lerner TN, Zeng H, 
            Zhu Y
  TITLE     Intersectional mapping of multi-transmitter neurons and other cell 
            types in the brain.
  JOURNAL   Cell Rep. 2022 Jul 5;40(1):111036. doi: 
            10.1016/j.celrep.2022.111036.
  PUBMED    35793636
REFERENCE   2  (bases 1 to 6311)
  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: "Cell Rep.";
            date: "2022-07-5"; volume: "40(1):111036. doi"; pages: " 
            10.1016/j.celrep.2022.111036"
COMMENT     Sequence Label: pAAV-EF1a-DIO-EGFP-WPRE
FEATURES             Location/Qualifiers
     source          1..6311
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        231..1409
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          462..1400
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1357..1377
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     protein_bind    complement(1440..1473)
                     /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(1524..1557)
                     /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(1566..2279,2280..2282,2283..2285))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1569..2279,2280..2282,2283..2285))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1611..1632)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1960..1979
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2219..2240
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2282..2291
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2296..2329
                     /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    2380..2413
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2438..3026
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2491..2511)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2909..2920)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3058..3534
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3257..3276)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3574..3714
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3574..3703
                     /label=AAV2 ITR
     rep_origin      3789..4244
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3876..3895)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4086..4107
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4261..4280)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4380..4402
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4440..4458)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4526..4630
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4631..4699,4700..5491)
                     /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(4849..4868)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5662..6250
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6151..6170
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaagctt tgcaaagatg gataaagttt taaacagaga
      181 ggaatctttg cagctaatgg accttctagg tcttgaaagg agtgggaatt ggctccggtg
      241 cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg
      301 gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt
      361 actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg
      421 tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc gtgtgtggtt
      481 cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt acttccacct
      541 ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg gagagttcga
      601 ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg cctgggcgct
      661 ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct ttcgataagt
      721 ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg caagatagtc
      781 ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc gcgggcggcg
      841 acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga gcgcggccac
      901 cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct ggcctcgcgc
      961 cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca gttgcgtgag
     1021 cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg acgcggcgct
     1081 cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg tcctcagccg
     1141 tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat tagttctcga
     1201 gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg gagtttcccc
     1261 acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa ttctccttgg
     1321 aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca gtggttcaaa
     1381 gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagtc gactccggaa
     1441 taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga ttgtagctgc
     1501 tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg aagttatggc
     1561 gcgccttact tgtacagctc gtccatgccg agagtgatcc cggcggcggt cacgaactcc
     1621 agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga ctgggtgctc
     1681 aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt ctgctggtag
     1741 tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa gttcaccttg
     1801 atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta gttgtactcc
     1861 agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag ctcgatgcgg
     1921 ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt gccgtcgtcc
     1981 ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt gaagaagtcg
     2041 tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt cagggtggtc
     2101 acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt cagggtcagc
     2161 ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg gccgtttacg
     2221 tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc gcccttgctc
     2281 accatggtgg ctagcataac ttcgtataaa gtatcctata cgaagttatt tgccttaacc
     2341 cagaaattat cactgttatt ctttagaatg gtgcaaagaa taacttcgta taatgtatgc
     2401 tatacgaagt tatgaattcg atatcaagct tatcgataat caacctctgg attacaaaat
     2461 ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc
     2521 tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
     2581 gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg
     2641 cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg
     2701 tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc
     2761 cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt
     2821 gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca cctggattct
     2881 gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg
     2941 cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg
     3001 gatctccctt tgggccgcct ccccgcatcg ataccgagcg ctgctcgaga gatctacggg
     3061 tggcatccct gtgacccctc cccagtgcct ctcctggccc tggaagttgc cactccagtg
     3121 cccaccagcc ttgtcctaat aaaattaagt tgcatcattt tgtctgacta ggtgtccttc
     3181 tataatatta tggggtggag gggggtggta tggagcaagg ggcaagttgg gaagacaacc
     3241 tgtagggcct gcggggtcta ttgggaacca agctggagtg cagtggcaca atcttggctc
     3301 actgcaatct ccgcctcctg ggttcaagcg attctcctgc ctcagcctcc cgagttgttg
     3361 ggattccagg catgcatgac caggctcagc taatttttgt ttttttggta gagacggggt
     3421 ttcaccatat tggccaggct ggtctccaac tcctaatctc aggtgatcta cccaccttgg
     3481 cctcccaaat tgctgggatt acaggcgtga accactgctc ccttccctgt ccttctgatt
     3541 ttgtaggtaa ccacgtgcgg accgagcggc cgcaggaacc cctagtgatg gagttggcca
     3601 ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc
     3661 cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagctgcctg caggggcgcc
     3721 tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca
     3781 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3841 cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct tcccttcctt
     3901 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3961 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg
     4021 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     4081 taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg gctattcttt
     4141 tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc tgatttaaca
     4201 aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag
     4261 tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga
     4321 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     4381 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg
     4441 cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc
     4501 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     4561 ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa
     4621 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
     4681 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
     4741 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
     4801 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
     4861 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
     4921 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
     4981 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
     5041 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
     5101 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
     5161 caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
     5221 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
     5281 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
     5341 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
     5401 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
     5461 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
     5521 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     5581 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     5641 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     5701 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     5761 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     5821 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     5881 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     5941 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     6001 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     6061 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     6121 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     6181 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     6241 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     6301 tgctcacatg t
//
