LOCUS       sequence-429396-        7740 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a Flag-tagged Rpl22I1 protein in cells expressing Cre 
            recombinase allowing for the harvesting of actively transcribed 
            mRNAs in that cell type..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7740)
  TITLE     GABAergic regulation of striatal spiny projection neuron depends 
            upon their activity state.
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7740)
  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            "Unpublished"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-hsyn-DIO-Rpl22l1-3Flag-2A-eGFP-WPRE
FEATURES             Location/Qualifiers
     source          1..7740
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             373..444
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     CDS             457..510
                     /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"
     CDS             join(511..513,514..516,517..1230)
                     /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             join(511..513,514..516,517..1227)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(556..577)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(817..836)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1164..1185
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    1239..1272
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    1323..1356
                     /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)."
     promoter        complement(1394..1841)
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     repeat_region   1886..2026
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1886..2015
                     /label=AAV2 ITR
     mobile_element  2081..2246
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      2430..2884
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2517..2536)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2726..2747
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2910..3014
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3015..3083,3084..3875)
                     /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(3233..3252)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4046..4634
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4535..4554
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     mobile_element  4939..5163
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(5230..5763)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLWSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(5933..5938,5939..5955,5956..5961))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     repeat_region   6301..6441
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   6312..6441
                     /label=AAV2 ITR
     polyA_signal    6481..6957
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     6739..6758
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     misc_feature    6989..7577
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             7095..7106
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     7504..7524
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(7602..7635)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(7686..7719)
                     /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)."
     regulatory      7726..7735
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 aaagacaaga agcctaagaa gtccacctgg aggtttcatt tggaccttac tcatccagta
       61 gaagatggaa tttttgactc tggaaatttt gaacagtttc tacgggagaa ggttaaagtc
      121 aatggaaaaa ctggaaatct tggcaatgtt gttcacattg aacgcctgaa gaataaaatc
      181 acagttgttt ctgagaaaca gttctccaaa aggtatttga aatatcttac caagaaatac
      241 cttaagaaga ataatctccg tgattggcta cgtgtggtcg catctgacaa ggagacttac
      301 gaacttcgtt atttccagat tagtcaagat gaagatggtt cagagtctga ggacacgcgt
      361 acgcggccgc tcgattacaa ggatgacgac gataaggact ataaggacga tgatgacaag
      421 gactacaaag atgatgacga taaatctggc agtggagagg gcagaggaag tctgctaaca
      481 tgcggtgacg tcgaggagaa tcctggccca atggtgagca agggcgagga gctgttcacc
      541 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
      601 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
      661 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag
      721 tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
      781 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
      841 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
      901 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac
      961 gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac
     1021 aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc
     1081 gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa
     1141 gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc
     1201 actctcggca tggacgagct gtacaagtaa ggcgcgccat aacttcgtat aatgtatgct
     1261 atacgaagtt attaagaggt ttcatattgc taatagcagc tacaatccag ctaccattct
     1321 gcataacttc gtataaagta tcctatacga agttattccg gagtcgactc tagaggatcc
     1381 ggtaccttct cgactgcgct ctcaggcacg acacgactcc tccgctgccc accgcagact
     1441 gaggcagcgc tgagtcgccg gcgccgcagc gcagatggtc gcgcccgtgc ccccctatct
     1501 cgcgcctcgc gtggtgcggt ccggctgggc cggcggcggc gcggacgcga ccaaggtggc
     1561 cgggaagggg agtttgcggg ggaccggcga gtgacgtcag cgcgccttca gtgctgaggc
     1621 ggcggtggcg cgcgccgcca ggcgggggcg aaggcactgt ccgcggtgct gaagctggca
     1681 gtgcgcacgc gcctcgccgc atcctgtttc ccctccccct ctctgatagg ggatgcgcaa
     1741 tttggggaat gggggttggg tgcttgtcca gtgggtcggg gtcggtcgtc aggtaggcac
     1801 ccccaccccg cctcatcctg gtcctaaaac ccacttgcac tcatacgcag ggccctctgc
     1861 agtctagaca cacgcgtgcg gccgcaggaa cccctagtga tggagttggc cactccctct
     1921 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     1981 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggtctg agacaataac
     2041 cctgataaat gcttcaataa tgtaagctct aggagatccg aaccagataa gtgaaatcta
     2101 gttccaaact attttgtcat ttttaatttt cgtattagct tacgacgcta cacccagttc
     2161 ccatctattt tgtcactctt ccctaaataa tccttaaaaa ctccatttcc acccctccca
     2221 gttcccaact attttgtccg cccacagcgg ggcatttttc ttcctgttat gtttttaatc
     2281 aaacatcctg ccaactccat gtgacaaacc gtcatcttcg gctacttttt ctctgtcaca
     2341 gaatgaaaat ttttctgtca tctcttcgtt attaatgttt gtaattgact gaatatcaac
     2401 gcttatttgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc
     2461 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2521 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2581 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2641 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2701 gacgttggag tccacgttct taatagtgga ctcttgttcc aaactggaac aacactcaac
     2761 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2821 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     2881 atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa
     2941 tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt
     3001 gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg
     3061 cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag
     3121 atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg
     3181 agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg
     3241 gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt
     3301 ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga
     3361 cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac
     3421 ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc
     3481 atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc
     3541 gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac
     3601 tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag
     3661 gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg
     3721 gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta
     3781 tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg
     3841 ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata
     3901 tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt
     3961 ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc
     4021 ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct
     4081 tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa
     4141 ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag
     4201 tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc
     4261 tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg
     4321 actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca
     4381 cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat
     4441 gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg
     4501 tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc
     4561 ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc
     4621 ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc
     4681 cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg
     4741 cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga
     4801 gcgaggaagc ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     4861 cacaccgcat agaccagccg cgtaacctgg caaaatcggt tacggttgag taataaatgg
     4921 atgccctgcg taagcgggtg tgggcggaca ataaagtctt aaactgaaca aaatagatct
     4981 aaactatgac aataaagtct taaactagac agaatagttg taaactgaaa tcagtccagt
     5041 tatgctgtga aaaagcatac tggacttttg ttatggctaa agcaaactct tcattttctg
     5101 aagtgcaaat tgcccgtcgt attaaagagg ggcgtggcca agggcatggt aaagactata
     5161 ttcgcggcgt tgtgacaatt taccgaacaa ctccgcggcc gggaagccga tctcggcttg
     5221 aacgaattgt taggtggcgg tacttgggtc gatatcaaag tgcatcactt cttcccgtat
     5281 gcccaacttt gtatagagag ccactgcggg atcgtcaccg taatctgctt gcacgtagat
     5341 cacataagca ccaagcgcgt tggcctcatg cttgaggaga ttgatgagcg cggtggcaat
     5401 gccctgcctc cggtgctcgc cggagactgc gagatcatag atatagatct cactacgcgg
     5461 ctgctcaaac ttgggcagaa cgtaagccgc gagagcgcca acaaccgctt cttggtcgaa
     5521 ggcagcaagc gcgatgaatg tcttactacg gagcaagttc ccgaggtaat cggagtccgg
     5581 ctgatgttgg gagtaggtgg ctacgtctcc gaactcacga ccgaaaagat caagagcagc
     5641 ccgcatggat ttgacttggt cagggccgag cctacatgtg cgaatgatgc ccatacttga
     5701 gccacctaac tttgttttag ggcgactgcc ctgctgcgta acatcgttgc tgctccataa
     5761 catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg gatgcccgag gcatagactg
     5821 tacaaaaaaa cagtcataac aagccatgaa aaccgccact gcgccgttac caccgctgcg
     5881 ttcggtcaag gttctggacc agttgcgtga gcgcatacgc tacttgcatt acagtttacg
     5941 aaccgaacag gcttatgtca actgggttcg tgccttcatc cgtttccacg gtgtgcgtca
     6001 cccggcaacc ttgggcagca gcgaagtcga ggcatttctg tcctggctgg cgaacgagcg
     6061 caaggtttcg gtctccacgc atcgtcaggc attggcggcc ttgctgttct tctacggcaa
     6121 ggtgctgtgc acggatctgc cctggcttca ggagatcgga agacctcggc cgtcgcggcg
     6181 cttgccggtg gtgctgaccc cggatgaagt ggttcgcatc ctcggttttc tggaaggcga
     6241 gcatcgtttg ttcgcccagg actctagcta tagttctagt ggttggctac agcttgcatg
     6301 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
     6361 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
     6421 actccatcac taggggttcc tgcggccgct cggtccgcac gtggttacct acaaaatcag
     6481 aaggacaggg aagggagcag tggttcacgc ctgtaatccc agcaatttgg gaggccaagg
     6541 tgggtagatc acctgagatt aggagttgga gaccagcctg gccaatatgg tgaaaccccg
     6601 tctctaccaa aaaaacaaaa attagctgag cctggtcatg catgcctgga atcccaacaa
     6661 ctcgggaggc tgaggcagga gaatcgcttg aacccaggag gcggagattg cagtgagcca
     6721 agattgtgcc actgcactcc agcttggttc ccaatagacc ccgcaggccc tacaggttgt
     6781 cttcccaact tgccccttgc tccataccac ccccctccac cccataatat tatagaagga
     6841 cacctagtca gacaaaatga tgcaacttaa ttttattagg acaaggctgg tgggcactgg
     6901 agtggcaact tccagggcca ggagaggcac tggggagggg tcacagggat gccacccgta
     6961 gatctctcga gcagcgctcg gtatcgatgc ggggaggcgg cccaaaggga gatccgactc
     7021 gtctgagggc gaaggcgaag acgcggaaga ggccgcagag ccggcagcag gccgcgggaa
     7081 ggaaggtccg ctggattgag ggccgaaggg acgtagcaga aggacgtccc gcgcagaatc
     7141 caggtggcaa cataggcgag cagccaagga aaggacgatg atttccccga caacaccacg
     7201 gaattgtcag tgcccaacag ccgagcccct gtccagcagc gggcaaggca ggcggcgatg
     7261 agttccgccg tggcaatagg gagggggaaa gcgaaagtcc cggaaaggag ctgacaggtg
     7321 gtggcaatgc cccaaccagt gggggttgcg tcagcaaaca cagtgcacac cacgccacgt
     7381 tgcctgacaa cgggccacaa ctcctcataa agagacagca accaggattt atacaaggag
     7441 gagaaaatga aagccatacg ggaagcaata gcatgataca aaggcattaa agcagcgtat
     7501 ccacatagcg taaaaggagc aacatagtta agaataccag tcaatctttc acaaattttg
     7561 taatccagag gttgattatc gataagcttg atatcgaatt cataacttcg tatagcatac
     7621 attatacgaa gttattcttt gcaccattct aaagaataac agtgataatt tctgggttaa
     7681 ggcaaataac ttcgtatagg atactttata cgaagttatg ctagcgccac catggcgccg
//
