LOCUS       sequence-441117-        7038 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent co-expression of FLAG-tagged inhibitory KORD 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 7038)
  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 7038)
  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: "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-KORD-P2A-ArgiNLS-AausFP1
FEATURES             Location/Qualifiers
     source          1..7038
                     /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(2366..2422)
                     /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             2927..2938
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             complement(3647..3670)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      3670..3679
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    3686..3719
                     /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    3770..3803
                     /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(3900..3920)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     repeat_region   4302..4442
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4302..4431
                     /label=AAV2 ITR
     rep_origin      4517..4972
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4604..4623)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4814..4835
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4989..5008)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5108..5130
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5168..5186)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5254..5358
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5359..5427,5428..6219)
                     /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(5577..5596)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6390..6977
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6878..6897
                     /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 ttcataggac cggggttttc ttccacgtct cctgcttgct
     2401 ttaacagaga gaagttcgtg gctccggatc ctactggttt attcatccca tcgatgtccc
     2461 tcaggtaagc aggatcctga actgtatttc ggactctgct agtgctctgc cgctccatcc
     2521 tcatcttcag tggaaagcag aagtcccgga aacaccgctt gaagttttca tcaagaaagg
     2581 cgtagagaat gggattcagg ctactgttgg tatagcctaa ggcgatgcag aagtaatagc
     2641 tggagagagc agctgtgctg tgggaggtgc tccccagagc ctccaccagg atgaatatgt
     2701 gaatgggagt ccagcagacg acgaagactg ccaccaccac caggaccagt ctggtgatcc
     2761 tacgcaggtt gcgatctttc tctcgggagc cagaaaggag ccggacgctc ttgagacgca
     2821 ggatcatcag ggtgtagcag acgatgatga tgaggacagg gatcacgaag gcaaagatga
     2881 agacgcagat cttcatgaag aggtcccacc aggagtagtc atcatctggg aactgcaagg
     2941 agcactcaat gacatcgacg tcttccctga ctttggtgcc tccaaggact attgcagaga
     3001 tgccaacaga tgacgacagc agccagatgc agatattgat gatctttgcc ttcaagggtg
     3061 tgcggaagtc caaagccttc acggggtggc acacggcaat gtagcggtcc acgctcatca
     3121 tggtcaaggt gaagatgctg gtgaacatgt tgtagtaatt aatggaaatt actatcttgc
     3181 acagcacatc cccaaaaggc caggaattca tcaagtagac cgtactctga aagggcatgg
     3241 ttgtagtaac taaagcatct gccaaagcca ggttaaatat gtaaatgttg gttgctgtct
     3301 tcatctttgt gtatcggatg atcacgaaca tgaccagcga gttgcccacc aagcccacga
     3361 cgaacactac ggagtagacc gccgtgatga tgaccgggat ggccggggag atgtgcgcgg
     3421 gctccagctg cgcgtcctcc gagccggcgc tgccgttgct gtcgggctcg gcccagccgg
     3481 gaaaccaggc gctgctgttg gggggcaggc aggcgctcgg ggcgcaggta gggcccggct
     3541 ccccgcggaa gatctgaatc ggggattcat ttaaatcttc ttctgataat aatttttgtt
     3601 ctaatgcatc tcgagtggat ccgagctctg aattctcacc gatatcttta tcatcatcat
     3661 ctttataatc catggtggcg ctagcataac ttcgtataaa gtatcctata cgaagttatt
     3721 tgccttaacc cagaaattat cactgttatt ctttagaatg gtgcaaagaa taacttcgta
     3781 taatgtatgc tatacgaagt tatgaattcg atatcaagct tatcgatatc gaattccgct
     3841 cgagataatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     3901 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     3961 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttagttct tgccacggcg
     4021 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     4081 aattccgtgg tgtttatttg tgaaatttgt gatgctattg ctttatttgt aaccatctag
     4141 ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa gctgcaataa
     4201 acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg agatgtggga
     4261 ggttttttaa agcggccgcc acgtgcggac cgagcggccg caggaacccc tagtgatgga
     4321 gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc
     4381 ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca
     4441 ggggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg
     4501 tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4561 acgcgcagcg tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc
     4621 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4681 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat
     4741 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     4801 acgttcttta atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc
     4861 tattcttttg atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg
     4921 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc
     4981 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     5041 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     5101 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     5161 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     5221 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     5281 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     5341 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     5401 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     5461 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     5521 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     5581 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     5641 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     5701 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     5761 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     5821 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     5881 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     5941 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     6001 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     6061 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     6121 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     6181 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     6241 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     6301 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     6361 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     6421 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     6481 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     6541 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     6601 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     6661 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac gggggttcgt
     6721 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     6781 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     6841 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     6901 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     6961 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7021 ggccttttgc tcacatgt
//
