LOCUS       sequence-486455-        7433 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Expression of synaptic GFP (no transcriptional control) and 
            cytosolic mRuby.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7433)
  TITLE     Dual FingR and Cytosolic Marker
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7433)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: EF1a-FLEX-Gephyrin.FR-GFP-p2A-mRuby
FEATURES             Location/Qualifiers
     source          1..7433
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     protein_bind    145..156
                     /label=ZF binding site
                     /bound_moiety="CCR5TC zinc-finger domain"
                     /note="target sequence for the CCR5TC zinc-finger domain 
                     from the CCR5-224 (+) zinc-finger nuclease (Perez et al., 
                     2008; Gross et al., 2013)"
     promoter        168..1346
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          399..1337
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1294..1314
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1363..1382
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1363..1381
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(1444..1477)
                     /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(1528..1561)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(1573..2283)
                     /codon_start=1
                     /product="monomeric red fluorescent protein derived from 
                     mRuby, with improved photophysical properties (Lam et al., 
                     2012)"
                     /label=mRuby2
                     /translation="MVSKGEELIKENMRMKVVMEGSVNGHQFKCTGEGEGNPYMGTQTM
                     RIKVIEGGPLPFAFDILATSFMYGSRTFIKYPKGIPDFFKQSFPEGFTWERVTRYEDGG
                     VVTVMQDTSLEDGCLVYHVQVRGVNFPSNGPVMQKKTKGWEPNTEMMYPADGGLRGYTH
                     MALKVDGGGHLSCSFVTTYRSKKTVGNIKMPGIHAVDHRLERLEESDNEMFVVQREHAV
                     AKFAGLGGGMDELYK"
     CDS             complement(2284..2340)
                     /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             join(2356..2358,2359..3066)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     CDS             join(2356..2358,2359..3066)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELY"
     primer_bind     complement(2398..2419)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2659..2678)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3006..3027
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             complement(3103..3126)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     protein_bind    3418..3451
                     /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    3502..3535
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3560..4148
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3613..3633)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4031..4042)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4180..4656
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(4379..4398)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4696..4836
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4696..4825
                     /label=AAV2 ITR
     rep_origin      4911..5366
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4998..5017)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5208..5229
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5383..5402)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5502..5524
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5562..5580)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5648..5752
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5753..5821,5822..6613)
                     /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(5971..5990)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6784..7372
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7273..7292
                     /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 gcgtgtcatc ctcatcgaat tccgtgaggc tccggtgccc
      181 gtcagtgggc agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca
      241 attgaaccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact
      301 ggctccgcct ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga
      361 acgttctttt tcgcaacggg tttgccgcca gaacacaggt aagtgccgtg tgtggttccc
      421 gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacctggc
      481 tgcagtacgt gattcttgat cccgagcttc gggttggaag tgggtgggag agttcgaggc
      541 cttgcgctta aggagcccct tcgcctcgtg cttgagttga ggcctggcct gggcgctggg
      601 gccgccgcgt gcgaatctgg tggcaccttc gcgcctgtct cgctgctttc gataagtctc
      661 tagccattta aaatttttga tgacctgctg cgacgctttt tttctggcaa gatagtcttg
      721 taaatgcggg ccaagatctg cacactggta tttcggtttt tggggccgcg ggcggcgacg
      781 gggcccgtgc gtcccagcgc acatgttcgg cgaggcgggg cctgcgagcg cggccaccga
      841 gaatcggacg ggggtagtct caagctggcc ggcctgctct ggtgcctggc ctcgcgccgc
      901 cgtgtatcgc cccgccctgg gcggcaaggc tggcccggtc ggcaccagtt gcgtgagcgg
      961 aaagatggcc gcttcccggc cctgctgcag ggagctcaaa atggaggacg cggcgctcgg
     1021 gagagcgggc gggtgagtca cccacacaaa ggaaaagggc ctttccgtcc tcagccgtcg
     1081 cttcatgtga ctccacggag taccgggcgc cgtccaggca cctcgattag ttctcgagct
     1141 tttggagtac gtcgtcttta ggttgggggg aggggtttta tgcgatggag tttccccaca
     1201 ctgagtgggt ggagactgaa gttaggccag cttggcactt gatgtaattc tccttggaat
     1261 ttgccctttt tgagtttgga tcttggttca ttctcaagcc tcagacagtg gttcaaagtt
     1321 tttttcttcc atttcaggtg tcgtgaggaa ttagcttggt actaatacga ctcactatag
     1381 ggagacccaa gctggctagg taagcttggt accgagctcg gatcctctag agtcgactcc
     1441 ggaataactt cgtataggat actttatacg aagttatgca gaatggtagc tggattgtag
     1501 ctgctattag caatatgaaa cctcttaata acttcgtata gcatacatta tacgaagtta
     1561 tggcgcgcct tacttgtaca gctcgtccat cccaccacca agcccggcga acttggcaac
     1621 tgcgtgttcg cgttgtacta cgaacatttc attgtcactt tcctctaacc tttccaggcg
     1681 gtgatcaacg gcatggatac cgggcatctt gatgttcccg acggtctttt ttgacctgta
     1741 agttgttacg aaagagcaag acagatggcc accaccatca actttcagtg ccatatgagt
     1801 gtatcccctc agaccaccat ctgctggata catcatctct gtattaggct cccaaccctt
     1861 ggtcttcttc tgcatcacgg gaccattgga gggaaagttt acccctctga cttggacgtg
     1921 gtaaacgaga cagccatcct caaggctggt gtcctgcatg acggtgacga ctccaccatc
     1981 ttcgtatctc gtaactcttt cccaagtaaa accctcagga aaggactgtt taaagaaatc
     2041 aggaatgcct ttcgggtact tgataaaagt acggctgcca tacatgaacg acgtggcaag
     2101 aatgtcaaag gcaaatggca ggggtcctcc ctcgatgact ttgatcctca tggtttgagt
     2161 tcccatgtac ggattgcctt ctccttcacc tgtgcatttg aattggtggc cgttgaccga
     2221 accttccatg accaccttca tacgcatatt ttccttgatc agctcttcgc ccttagacac
     2281 catgggaccg gggttttctt ccacgtctcc tgcttgcttt aacagagaga agttcgtggc
     2341 tccggagccc acaccgtgag caagggcgag gagctgttca ccggggtggt gcccatcctg
     2401 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc
     2461 gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
     2521 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc
     2581 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
     2641 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag
     2701 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac
     2761 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac
     2821 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
     2881 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg
     2941 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
     3001 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag
     3061 ctgtacagta ccacctgaac ctccagtacc acctgaacct cccttatcgt cgtcatcctt
     3121 gtaatcggat ccggtgcggt agttgatgga gatcgggatc gggtcctggt agttccagtg
     3181 gtcgatcgtg acggcgtaca ccgtgatggt atagtcgaca ccaggtttca ggctgctgat
     3241 ggtagcagtg gacttgctgc caggcacggt gaattcctgg acagggctat tgccaccagt
     3301 ttcaccgtag gtgatgcggt agtagcgaac catgaccttg tacttgcccc agctgatctg
     3361 gatgctggtt ggtgatgctt ccttgacttc gagcatggtg gcggcggtgg cgctagcata
     3421 acttcgtata aagtatccta tacgaagtta tttgccttaa cccagaaatt atcactgtta
     3481 ttctttagaa tggtgcaaag aataacttcg tataatgtat gctatacgaa gttatgaatt
     3541 cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa gattgactgg
     3601 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta
     3661 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct
     3721 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt
     3781 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac
     3841 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg
     3901 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc
     3961 gtcctttcct tggctgctcg cctatgttgc cacctggatt ctgcgcggga cgtccttctg
     4021 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct
     4081 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc
     4141 ctccccgcat cgataccgag cgctgctcga gagatctacg ggtggcatcc ctgtgacccc
     4201 tccccagtgc ctctcctggc cctggaagtt gccactccag tgcccaccag ccttgtccta
     4261 ataaaattaa gttgcatcat tttgtctgac taggtgtcct tctataatat tatggggtgg
     4321 aggggggtgg tatggagcaa ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc
     4381 tattgggaac caagctggag tgcagtggca caatcttggc tcactgcaat ctccgcctcc
     4441 tgggttcaag cgattctcct gcctcagcct cccgagttgt tgggattcca ggcatgcatg
     4501 accaggctca gctaattttt gtttttttgg tagagacggg gtttcaccat attggccagg
     4561 ctggtctcca actcctaatc tcaggtgatc tacccacctt ggcctcccaa attgctggga
     4621 ttacaggcgt gaaccactgc tcccttccct gtccttctga ttttgtaggt aaccacgtgc
     4681 ggaccgagcg gccgcaggaa cccctagtga tggagttggc cactccctct ctgcgcgctc
     4741 gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt gcccgggcgg
     4801 cctcagtgag cgagcgagcg cgcagctgcc tgcaggggcg cctgatgcgg tattttctcc
     4861 ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt acgcgccctg
     4921 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4981 cagcgcctta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     5041 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     5101 gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc catcgccctg
     5161 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     5221 ccaaactgga acaacactca actctatctc gggctattct tttgatttat aagggatttt
     5281 gccgatttcg gtctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     5341 taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct gctctgatgc
     5401 cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5461 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5521 gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt
     5581 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     5641 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     5701 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     5761 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     5821 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     5881 ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     5941 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga
     6001 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     6061 ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6121 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6181 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6241 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc
     6301 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6361 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6421 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6481 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6541 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     6601 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     6661 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     6721 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     6781 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     6841 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     6901 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     6961 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     7021 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     7081 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     7141 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     7201 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7261 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7321 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7381 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgt
//
