LOCUS       sequence-343527-        7005 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Constitutive expression of EBFP and rtTA-InteinN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7005)
  AUTHORS   Chen HS, Zhang XL, Yang RR, Wang GL, Zhu XY, Xu YF, Wang DY, Zhang 
            N, Qiu S, Zhan LJ, Shen ZM, Xu XH, Long G, Xu C
  TITLE     An intein-split transactivator for intersectional neural imaging and
            optogenetic manipulation.
  JOURNAL   Nat Commun. 2022 Jun 23;13(1):3605. doi: 10.1038/s41467-022-31255-x.
  PUBMED    35739125
REFERENCE   2  (bases 1 to 7005)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2022-06-23"; volume: "13(1):3605. doi"; pages: " 
            10.1038/s41467-022-31255-x"
COMMENT     Sequence Label: pAAV-pEF1a-EBFP-tTAn
FEATURES             Location/Qualifiers
     source          1..7005
                     /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"
     regulatory      1428..1437
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1434..1436,1437..1439,1440..2150)
                     /codon_start=1
                     /product="enhanced blue variant of GFP (Ai et al., 2007)"
                     /label=EBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTLSHGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGVDFKEDGNILGHKLEYNFNSHNIYIMAVKQKNGIK
                     VNFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDSHYLSTQSVLSKDPNEKRDHMVLL
                     EFRTAAGITLGMDELYK"
     primer_bind     complement(1479..1500)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             2160..2213
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     misc_feature    3132..3720
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3185..3205)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3603..3614)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3752..4228
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3951..3970)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4268..4408
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4268..4397
                     /label=AAV2 ITR
     rep_origin      4483..4938
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4570..4589)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4780..4801
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4955..4974)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5074..5096
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5134..5152)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5220..5324
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5325..5393,5394..6185)
                     /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(5543..5562)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6356..6944
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6845..6864
                     /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 ctctagagcc accatggtga
     1441 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     1501 taaacggcca caagttcagc gtgaggggcg agggcgaggg cgatgccacc aacggcaagc
     1561 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1621 ccaccctgag ccacggcgtg cagtgcttcg cccgctaccc cgaccacatg aagcagcacg
     1681 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     1741 acgacggcac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctagtgaacc
     1801 gcatcgagct gaagggcgtc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1861 agtacaactt caacagccac aacatctata tcatggccgt caagcagaag aacggcatca
     1921 aggtgaactt caagatccgc cacaacgtgg aggacggcag cgtgcagctc gccgaccact
     1981 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacagc cactacctga
     2041 gcacccagtc cgtgctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2101 agttccgcac cgccgccggg atcactctcg gcatggacga gctgtacaag ggcagtggag
     2161 agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccatctagac
     2221 tggacaagag caaagtcata aactctgctc tggaattact caatgaagtc ggtatcgaag
     2281 gcctgacgac aaggaaactc gctcaaaagc tgggagttga gcagcctacc ctgtactggc
     2341 acgtgaagaa caagcgggcc ctgctcgatg ccctggcaat cgagatgctg gacaggcatc
     2401 atacccactt ctgccccctg gaaggcgagt catggcaaga ctttctgcgg aacaacgcca
     2461 agtcattccg ctgtgctctc ctctcacatc gcgacggggc taaagtgcat ctcggcaccc
     2521 gcccaacaga gaaacagtac gaaaccctgg aaaatcagct cgcgttcctg tgtcagcaag
     2581 gcttctccct ggagaacgca ctgtacgctc tgtccgccgt gggccacttt acactgggct
     2641 gcgtattgga ggatcaggag catcaagtag caaaagagga aagagagaca cctaccaccg
     2701 attctatgcc cccacttctg agacaagcaa ttgagctgtt cgaccatcag ggagccgaac
     2761 ctgccttcct tttcggcctg gaactaatca tatgtggcct ggagaaacag ctaaagtgcg
     2821 aaagcggcgg gacccgcagc ggctactgcc tggacctgaa gacccaggtg cagacccccc
     2881 agggcatgaa ggagatcagc aacatccagg tgggcgacct ggtgctgagc aacaccggct
     2941 acaacgaggt gctgaacgtg ttccccaaga gcaagaagaa gagctacaag atcaccctgg
     3001 aggacggcaa ggagatcatc tgcagcgagg agcacctgtt ccccacccag accggcgaga
     3061 tgaacatcag cggcggcctg aaggagggca tgtgcctgta cgtgaaggag taagatatca
     3121 agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta
     3181 actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta
     3241 ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt
     3301 atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg
     3361 caacccccac tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt
     3421 tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag
     3481 gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc
     3541 cttggctgct cgcctatgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc
     3601 cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc
     3661 ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc
     3721 atcgataccg agcgctgctc gagagatcta cgggtggcat ccctgtgacc cctccccagt
     3781 gcctctcctg gccctggaag ttgccactcc agtgcccacc agccttgtcc taataaaatt
     3841 aagttgcatc attttgtctg actaggtgtc cttctataat attatggggt ggaggggggt
     3901 ggtatggagc aaggggcaag ttgggaagac aacctgtagg gcctgcgggg tctattggga
     3961 accaagctgg agtgcagtgg cacaatcttg gctcactgca atctccgcct cctgggttca
     4021 agcgattctc ctgcctcagc ctcccgagtt gttgggattc caggcatgca tgaccaggct
     4081 cagctaattt ttgttttttt ggtagagacg gggtttcacc atattggcca ggctggtctc
     4141 caactcctaa tctcaggtga tctacccacc ttggcctccc aaattgctgg gattacaggc
     4201 gtgaaccact gctcccttcc ctgtccttct gattttgtag gtaaccacgt gcggaccgag
     4261 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc
     4321 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg
     4381 agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct ccttacgcat
     4441 ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc tgtagcggcg
     4501 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgcct
     4561 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
     4621 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
     4681 accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
     4741 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
     4801 gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt ttgccgattt
     4861 cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
     4921 tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     4981 taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     5041 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     5101 caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg
     5161 ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
     5221 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     5281 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     5341 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     5401 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     5461 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     5521 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     5581 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     5641 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     5701 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     5761 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     5821 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     5881 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     5941 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     6001 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     6061 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     6121 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     6181 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     6241 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     6301 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     6361 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     6421 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     6481 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     6541 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     6601 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     6661 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     6721 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     6781 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     6841 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     6901 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6961 cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
