LOCUS       sequence-237593-        7089 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  An AAV vector that expresses a nuclear-localized iRFP713 reporter 
            and optimized Flp recombinase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7089)
  AUTHORS   Back S, Necarsulmer J, Whitaker LR, Coke LM, Koivula P, Heathward 
            EJ, Fortuno LV, Zhang Y, Yeh CG, Baldwin HA, Spencer MD, 
            Mejias-Aponte CA, Pickel J, Hoffman AF, Spivak CE, Lupica CR, 
            Underhill SM, Amara SG, Domanskyi A, Anttila JE, Airavaara M, Hope 
            BT, Hamra FK, Richie CT, Harvey BK
  TITLE     Neuron-Specific Genome Modification in the Adult Rat Brain Using 
            CRISPR-Cas9 Transgenic Rats.
  JOURNAL   Neuron. 2019 Feb 8. pii: S0896-6273(19)30062-5. doi: 
            10.1016/j.neuron.2019.01.035.
  PUBMED    30792150
REFERENCE   2  (bases 1 to 7089)
  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: "Neuron. 
            2019 Feb 8. pii: S0896-6273(19)30062-5. doi: 
            10.1016/j.neuron.2019.01.035."
COMMENT     Sequence Label: pOTTC1440 - pAAV CMV-IE Nuc-iRFP-2A-Flpo
FEATURES             Location/Qualifiers
     source          1..7089
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     enhancer        205..508
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        509..712
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     663..683
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      747..756
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             753..1700
                     /codon_start=1
                     /product="phytochrome-based near-infrared fluorescent 
                     protein, originally termed iRFP (Filonov et al., 2011)"
                     /label=iRFP713
                     /note="name was changed to iRFP713 (Shcherbakova and 
                     Verkhusha, 2013)"
                     /translation="MAEGSVARQPDLLTCDDEPIHIPGAIQPHGLLLALAADMTIVAGS
                     DNLPELTGLAIGALIGRSAADVFDSETHNRLTIALAEPGAAVGAPITVGFTMRKDAGFI
                     GSWHRHDQLIFLELEPPQRDVAEPQAFFRRTNSAIRRLQAAETLESACAAAAQEVRKIT
                     GFDRVMIYRFASDFSGEVIAEDRCAEVESKLGLHYPASTVPAQARRLYTINPVRIIPDI
                     NYRPVPVTPDLNPVTGRPIDLSFAILRSVSPVHLEFMRNIGMHGTMSISILRGERLWGL
                     IVCHHRTPYYVDLDGRQACELVAQVLAWQIGVMEE"
     CDS             1734..1754
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1758..1778
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1782..1802
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1833..1886
                     /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(1893..1898,1899..1919,1920..3191)
                     /codon_start=1
                     /product="nuclear-targeted site-specific recombinase"
                     /label=FLPo
                     /note="FLPo is a mouse codon-optimized fusion between the 
                     SV40 nuclear localization signal and a thermostable version
                     of the Saccharomyces cerevisiae site-specific recombinase 
                     FLP.  Recombination occurs at FRT sequences."
                     /translation="MAPKKKRKVMSQFDILCKTPPKVLVRQFVERFERPSGEKIASCAA
                     ELTYLCWMITHNGTAIKRATFMSYNTIISNSLSFDIVNKSLQFKYKTQKATILEASLKK
                     LIPAWEFTIIPYNGQKHQSDITDIVSSLQLQFESSEEADKGNSHSKKMLKALLSEGESI
                     WEITEKILNSFEYTSRFTKTKTLYQFLFLATFINCGRFSDIKNVDPKSFKLVQNKYLGV
                     IIQCLVTETKTSVSRHIYFFSARGRIDPLVYLDEFLRNSEPVLKRVNRTGNSSSNKQEY
                     QLLKDNLVRSYNKALKKNAPYPIFAIKNGPKSHIGRHLMTSFLSMKGLTELTNVVGNWS
                     DKRASAVARTTYTHQITAIPDHYFALVSRYYAYDPISKEMIALKDETNPIEEWQHIEQL
                     KGSAEGSIRYPAWNGIISQEVLDYLSSYINRRI"
     misc_feature    3216..3804
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3269..3289)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3687..3698)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3836..4312
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4035..4054)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4352..4492
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4352..4481
                     /label=AAV2 ITR
     rep_origin      4567..5022
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4654..4673)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4864..4885
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5039..5058)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5158..5180
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5218..5236)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5304..5408
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5409..5477,5478..6269)
                     /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(5627..5646)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6440..7028
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6929..6948
                     /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 gcgttagtta ttaatagtaa tcaattacgg ggtcattagt
      181 tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg
      241 accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc
      301 aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc
      361 agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg
      421 gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat
      481 ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg
      541 tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag
      601 tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt
      661 gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt
      721 gaaccgtcag atccggtacc ggatccgcca ccatggctga aggatccgtc gccaggcagc
      781 ctgacctctt gacctgcgac gatgagccga tccatatccc cggtgccatc caaccgcatg
      841 gactgctgct cgccctcgcc gccgacatga cgatcgttgc cggcagcgac aaccttcccg
      901 aactcaccgg actggcgatc ggcgccctga tcggccgctc tgcggccgat gtcttcgact
      961 cggagacgca caaccgtctg acgatcgcct tggccgagcc cggggcggcc gtcggagcac
     1021 cgatcactgt cggcttcacg atgcgaaagg acgcaggctt catcggctcc tggcatcgcc
     1081 atgatcagct catcttcctc gagctcgagc ctccccagcg ggacgtcgcc gagccgcagg
     1141 cgttcttccg ccgcaccaac agcgccatcc gccgcctgca ggccgccgaa accttggaaa
     1201 gcgcctgcgc cgccgcggcg caagaggtgc ggaagattac cggcttcgat cgggtgatga
     1261 tctatcgctt cgcctccgac ttcagcggcg aagtgatcgc agaggatcgg tgcgccgagg
     1321 tcgagtcaaa actaggcctg cactatcctg cctcaaccgt gccggcgcag gcccgtcggc
     1381 tctataccat caacccggta cggatcattc ccgatatcaa ttatcggccg gtgccggtca
     1441 ccccagacct caatccggtc accgggcggc cgattgatct tagcttcgcc atcctgcgca
     1501 gcgtctcgcc cgtccatctg gaattcatgc gcaacatagg catgcacggc acgatgtcga
     1561 tctcgatttt gcgcggcgag cgactgtggg gattgatcgt ttgccatcac cgaacgccgt
     1621 actacgtcga tctcgatggc cgccaagcct gcgagctagt cgcccaggtt ctggcctggc
     1681 agatcggcgt gatggaagag ttgtacaagt ccggcctgcg cagccgcgcc gacccaaaaa
     1741 agaagcgcaa ggtagacccc aagaagaagc gcaaggtaga tccaaaaaag aagagaaagg
     1801 tactgtacaa gatgcattct agaggctccg gagagggcag gggaagtctt ctaacatgcg
     1861 gggacgtgga ggaaaatccc gggcccatcg atatggctcc taagaagaag aggaaggtga
     1921 tgagccagtt cgacatcctg tgcaagaccc cccccaaggt gctggtgcgg cagttcgtgg
     1981 agagattcga gaggcccagc ggcgagaaga tcgccagctg tgccgccgag ctgacctacc
     2041 tgtgctggat gatcacccac aacggcaccg ccatcaagag ggccaccttc atgagctaca
     2101 acaccatcat cagcaacagc ctgagcttcg acatcgtgaa caagagcctg cagttcaagt
     2161 acaagaccca gaaggccacc atcctggagg ccagcctgaa gaagctgatc cccgcctggg
     2221 agttcaccat catcccttac aacggccaga agcaccagag cgacatcacc gacatcgtgt
     2281 ccagcctgca gctgcagttc gagagcagcg aggaggccga caagggcaac agccacagca
     2341 agaagatgct gaaggccctg ctgtccgagg gcgagagcat ctgggagatc accgagaaga
     2401 tcctgaacag cttcgagtac accagcaggt tcaccaagac caagaccctg taccagttcc
     2461 tgttcctggc cacattcatc aactgcggca ggttcagcga catcaagaac gtggacccca
     2521 agagcttcaa gctggtgcag aacaagtacc tgggcgtgat cattcagtgc ctggtgaccg
     2581 agaccaagac aagcgtgtcc aggcacatct actttttcag cgccagaggc aggatcgacc
     2641 ccctggtgta cctggacgag ttcctgagga acagcgagcc cgtgctgaag agagtgaaca
     2701 ggaccggcaa cagcagcagc aacaagcagg agtaccagct gctgaaggac aacctggtgc
     2761 gcagctacaa caaggccctg aagaagaacg ccccctaccc catcttcgct atcaagaacg
     2821 gccctaagag ccacatcggc aggcacctga tgaccagctt tctgagcatg aagggcctga
     2881 ccgagctgac aaacgtggtg ggcaactgga gcgacaagag ggcctccgcc gtggccagga
     2941 ccacctacac ccaccagatc accgccatcc ccgaccacta cttcgccctg gtgtccaggt
     3001 actacgccta cgaccccatc agcaaggaga tgatcgccct gaaggacgag accaacccca
     3061 tcgaggagtg gcagcacatc gagcagctga agggcagcgc cgagggcagc atcagatacc
     3121 ccgcctggaa cggcatcatc agccaggagg tgctggacta cctgagcagc tacatcaaca
     3181 ggcggatctg agaattcgat atcaagctta tcgataatca acctctggat tacaaaattt
     3241 gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg
     3301 ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt
     3361 ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg
     3421 tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc
     3481 agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg
     3541 cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt
     3601 tgtcggggaa atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc
     3661 gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg
     3721 gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga
     3781 tctccctttg ggccgcctcc ccgcatcgat accgagcgct gctcgagaga tctacgggtg
     3841 gcatccctgt gacccctccc cagtgcctct cctggccctg gaagttgcca ctccagtgcc
     3901 caccagcctt gtcctaataa aattaagttg catcattttg tctgactagg tgtccttcta
     3961 taatattatg gggtggaggg gggtggtatg gagcaagggg caagttggga agacaacctg
     4021 tagggcctgc ggggtctatt gggaaccaag ctggagtgca gtggcacaat cttggctcac
     4081 tgcaatctcc gcctcctggg ttcaagcgat tctcctgcct cagcctcccg agttgttggg
     4141 attccaggca tgcatgacca ggctcagcta atttttgttt ttttggtaga gacggggttt
     4201 caccatattg gccaggctgg tctccaactc ctaatctcag gtgatctacc caccttggcc
     4261 tcccaaattg ctgggattac aggcgtgaac cactgctccc ttccctgtcc ttctgatttt
     4321 gtaggtaacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact
     4381 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg
     4441 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg
     4501 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatacg tcaaagcaac
     4561 catagtacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg
     4621 tgaccgctac acttgccagc gccttagcgc ccgctccttt cgctttcttc ccttcctttc
     4681 tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc
     4741 gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta
     4801 gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta
     4861 atagtggact cttgttccaa actggaacaa cactcaactc tatctcgggc tattcttttg
     4921 atttataagg gattttgccg atttcggtct attggttaaa aaatgagctg atttaacaaa
     4981 aatttaacgc gaattttaac aaaatattaa cgtttacaat tttatggtgc actctcagta
     5041 caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg
     5101 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg
     5161 ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc
     5221 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag
     5281 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt
     5341 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa
     5401 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt
     5461 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt
     5521 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt
     5581 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg
     5641 tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga
     5701 atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa
     5761 gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga
     5821 caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa
     5881 ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca
     5941 ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta
     6001 ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac
     6061 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
     6121 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
     6181 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
     6241 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
     6301 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     6361 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     6421 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
     6481 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
     6541 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
     6601 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
     6661 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
     6721 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
     6781 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
     6841 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
     6901 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
     6961 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
     7021 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     7081 ctcacatgt
//
