LOCUS       sequence-441115-        7347 bp    DNA     circular SYN 02-JAN-2026
DEFINITION  Cre-dependent co-expression of FLAG-tagged inhibitory hM4D(Gi) 
            DREADD receptor, and a single-cell discriminating version of AausFP1
            as a reporter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7347)
  AUTHORS   Szelenyi ER, Navarrete JS, Murry AD, Zhang Y, Girven KS, Kuo L, 
            Cline MM, Bernstein MX, Burdyniuk M, Bowler B, Goodwin NL, Juarez B,
            Zweifel LS, Golden SA
  TITLE     An arginine-rich nuclear localization signal (ArgiNLS) strategy for 
            streamlined image segmentation of single cells.
  JOURNAL   Proc Natl Acad Sci U S A. 2024 Aug 6;121(32):e2320250121. doi: 
            10.1073/pnas.2320250121. Epub 2024 Jul 29.
  PUBMED    39074275
REFERENCE   2  (bases 1 to 7347)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 2, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc Natl 
            Acad Sci U S A."; date: "2024-08-6"; volume: "121(32):e2320250121. 
            doi"; pages: " 10.1073/pnas.2320250121. Epub 2024 Jul 29"
COMMENT     Sequence Label: pAAV-EF1a-DIO-FLAG-hM4D(Gi)-P2A-ArgiNLS-AausFP1
FEATURES             Location/Qualifiers
     source          1..7347
                     /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(1445..1478)
                     /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(1529..1562)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2363..2419)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(3956..3979)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3979..3988
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3995..4028
                     /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    4079..4112
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4209..4229)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4611..4751
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4611..4740
                     /label=AAV2 ITR
     rep_origin      4826..5281
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4913..4932)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5123..5144
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5298..5317)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5417..5439
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5477..5495)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5563..5667
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5668..5736,5737..6528)
                     /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(5886..5905)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6699..7286
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7187..7206
                     /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 gtaccgtacc ggatcctcta gagtcgactc
     1441 cggaataact tcgtatagga tactttatac gaagttatgc agaatggtag ctggattgta
     1501 gctgctatta gcaatatgaa acctcttaat aacttcgtat agcatacatt atacgaagtt
     1561 atggcgcgcc ttaggcgtag gcggtcttgc agtcgatggc tctgaacact tccaccacgt
     1621 tcatgtggtc ccgggtttct ttagggtcct tgctcaggat ggtgtgggcc tgaatgtggt
     1681 gatagtgagg gatgtccacg gggccacctc cgagaggggt attcagctgc tggtgagcag
     1741 caatgatctg gcctccgccg atcacgtcgt gcacaatgtt gatgtagcac ttgatgccgt
     1801 tgttctgcac gtcgggcagc acgtagatgc agtgcgggtt gtagttgaac tcaagtttct
     1861 tgcccaggat gttgccgcct ttcttgaagc cgctgccgtt cagggtcact ctgttgtaca
     1921 cggagccctt ctcgtaggtc acttcggctc tggtcttgta cacgccgtcg ttctcgaagg
     1981 tgatggttct ctcttgcacg tagccctcag gcatggcgct cttggggtag tccttgatgt
     2041 cgttgggata cttggcgaag cactgggcgc cgtatgtcac tgtggtcagg atgctgctcc
     2101 aaggcacagg cagttcgccg gttgtgcaga tgaagctggc ctcgatcttt ccggtattgg
     2161 cgtcgccgtg tcctttgcct ctgacgctga acttcatgcc ttccacgtcg ccttccattt
     2221 ccaccacata ggggatcttc tcgcggaaca gcagggcgcc ataagacatg gtggccacag
     2281 gggatcccgt tcgacggcgc cgccgccttc tcaccttctt tttctttgct atgctcatct
     2341 gcttctgggg agccaccctt ttaggaccgg ggttttcttc cacgtctcct gcttgcttta
     2401 acagagagaa gttcgtggct ccggatccga ccggtggatc ccgcctggca gtgccgatgt
     2461 tccgatactg gcacagcagc aggtgccgga aggtcttttt aaaggtggcg ttgcacagag
     2521 catagcaggc agggttgatg gtgctgttga cgtagcagag ccagtagcca atggaccaca
     2581 ccgtgtcagg gatgcagctc tggcagaagg tgttcaccag gaccatgacg ttgtagggcg
     2641 tccaggtgag gatgaaggcc agcagaatgg caaagatcgt tcgtgtcact ttgcgctccc
     2701 gggccgccat ctgccgcttc ttgcgcacct ggttgcgagc gatgctggcg aacttgcggg
     2761 ccacgttggc cgcagggcgc atgccagccg gcgtggcagg cacaatctca atggctgtca
     2821 cacactcatt gcctgtctgc ttcgtcacaa tctggatctt ggaccatctg gaggctgggt
     2881 tgagggcccg cggctgcagg ggaggggcgg gcatggcggg cgtggtggcc tctgtggtgg
     2941 acagctctgt ggctgggcgt tccttggtgt tctgggtggc actgcctgag ctggactcat
     3001 tggaagtgtc cttatcagcc acggggcgcg gtggcggtgg cagcgctggc gggggggcct
     3061 cctccagctt gccattgcgc agctcctccc gggcggcctc cccgggcggg ggcttcttga
     3121 cgctctgctt cattagtggg ctcttgagga aggccagcgt cttggctttc ttctccttcg
     3181 ggccctcggg ccggtgcttg tggactcggc tgcgactggc cagggagatg tggatgtaca
     3241 gcaccgtcat gatgaccaca ggcaggtaga agccagcaat ggctgtgcca aaggtcactg
     3301 ctgggttgga caggaactgg atgaagcact ggttgtcggg caccgtccgc ttacccacca
     3361 caaactgcca gaacaagatg gcaggcgccc agagcacgaa ggacagtacc caggcagcag
     3421 caatcatgag gcctgccatc ttggtggtgc gccgggcagg gtaggtgaga ggcttggtga
     3481 cgcagaagta gcggtcaaag ctgatgatga gaaggttcat gacggaggcg ttgctcacca
     3541 cgcagtccag ggccagccac aggtcgcaga ccacggcgcc caggggccag tagcccttga
     3601 tgatgtacac ggtgtagagg ttcatggaga aggcgcctat gatgagatca gcacacgcca
     3661 ggctgaagag gaagtagttg ttgactgtct gcagctgcct gttgaccttg atggacagca
     3721 tcaccaggat gttgcccacg acagtcacca ggctcaggga gcctgtcact gtggcaatga
     3781 agaccatttc caccgtctca tagcgattgt gggatgatga cgtgaccagg cgcacggact
     3841 gattgcccga gctgccattg acaggtgtga agttggcatt taaatcttct tctgataata
     3901 atttttgttc taatgcatct cgagtggatc cgagctctga attctcaccg atatctttat
     3961 catcatcatc tttataatcc atggtggcgc tagcataact tcgtataaag tatcctatac
     4021 gaagttattt gccttaaccc agaaattatc actgttattc tttagaatgg tgcaaagaat
     4081 aacttcgtat aatgtatgct atacgaagtt atgaattcga tatcaagctt atcgatatcg
     4141 aattccgctc gagataatca acctctggat tacaaaattt gtgaaagatt gactggtatt
     4201 cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat
     4261 gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttagttctt
     4321 gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg
     4381 ggcactgaca attccgtggt gtttatttgt gaaatttgtg atgctattgc tttatttgta
     4441 accatctagc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag
     4501 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga
     4561 gatgtgggag gttttttaaa gcggccgcca cgtgcggacc gagcggccgc aggaacccct
     4621 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     4681 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     4741 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     4801 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     4861 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     4921 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     4981 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5041 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5101 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     5161 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     5221 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5281 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5341 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5401 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5461 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5521 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5581 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     5641 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     5701 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     5761 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     5821 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     5881 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     5941 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     6001 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6061 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6121 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6181 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6241 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6301 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6361 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6421 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6481 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6541 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6601 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     6661 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6721 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6781 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6841 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6901 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6961 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     7021 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     7081 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     7141 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     7201 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     7261 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     7321 ccttttgctg gccttttgct cacatgt
//
