LOCUS       sequence-12316-e        6912 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6912)
  AUTHORS   Wall NR, Wickersham IR, Cetin A, De La Parra M, Callaway EM
  TITLE     Monosynaptic circuit tracing in vivo through Cre-dependent targeting
            and complementation of modified rabies virus.
  JOURNAL   Proc Natl Acad Sci U S A. 2010 Dec 14;107(50):21848-53. doi: 
            10.1073/pnas.1011756107
  PUBMED    21115815
REFERENCE   2  (bases 1 to 6912)
  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"; doi: "10.1073/pnas"; 
            journalName: "Proc Natl Acad Sci U S A"; date: "2010-12-14- 14"; 
            volume: "107"; issue: "50"; pages: "21848-53"
COMMENT     Sequence Label: pAAV-EF1a-FLEX-GT
FEATURES             Location/Qualifiers
     source          1..6912
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     promoter        242..1419
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          473..1410
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1367..1387
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1441..1450
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    1453..1486
                     /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    1549..1582
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(2069..2122)
                     /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             complement(join(2138..2848,2849..2851))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(2138..2848,2849..2851))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2180..2201)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2529..2548
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2788..2809
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    complement(2860..2893)
                     /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(2964..2997)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    3039..3627
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3092..3112)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3510..3521)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3659..4135
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3858..3877)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   4175..4315
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4175..4304
                     /label=AAV2 ITR
     rep_origin      4390..4845
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4477..4496)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4687..4708
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4862..4881)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4981..5003
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5041..5059)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5127..5231
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5232..5300,5301..6092)
                     /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(5450..5469)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6263..6851
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6752..6771
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc tgcggccgca cgcgtaagct ttgcaaagat ggataaagtt
      181 ttaaacagag aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
      241 tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg
      301 gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag
      361 tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc
      421 agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc
      481 cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
      541 tacttccact ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      601 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      661 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      721 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      781 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      841 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      901 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      961 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     1021 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     1081 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     1141 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     1201 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1261 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1321 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1381 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gtaccggatc ctctagagcc
     1441 gccaccatgg ccataacttc gtataaagta tcctatacga agttatatca aaataggaag
     1501 accaatgctt caccatcgac ccgaattgcc aagcatcacc atcgacccat aacttcgtat
     1561 aatgtatgct atacgaagtt atacttcaac tagcggagaa caagtctgcc tggctcttgt
     1621 caggcaccag cagctccttg gatgcgcttt caagactgaa gatgtcagac ctgctttttg
     1681 ctttggactt cccccatgca gcgataccac ccacagctac caggcagcac aggagcactg
     1741 cagtgatcag catccacatg cggccgtgat tcctggctgg cagagcacgt ccaggagcag
     1801 gggcagtggg agcctctgtg ccgttgtccg tgggcaccgc ggggatcgcg ctggtcccgc
     1861 agccccactc gtcccgcccg tcgtcgcagt cggggtgtcc gtcgcacagc cagtcctgcg
     1921 ggtaacactc cccgtgggca ccgggcggct ccgagcagcg gaactgaccg ggggggcaac
     1981 gggacaaaga accgttaccg gacccgttac cggtcacgtt accgggcagc agcagcagca
     2041 gcagcgcggg cagcagccgc gcatcgattg ggccaggatt ctcctcgacg tcaccgcatg
     2101 ttagcagact tcctctgccc tctccactac caagcttctt gtacagctcg tccatgccga
     2161 gagtgatccc ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg
     2221 ggtctttgct cagggcggac tgggtgctca ggtagtggtt gtcgggcagc agcacggggc
     2281 cgtcgccgat gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga
     2341 tgttgtggcg gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg gccatgatat
     2401 agacgttgtg gctgttgtag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct
     2461 tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct
     2521 cggcgcgggt cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc
     2581 cttcgggcat ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcggctga
     2641 agcactgcac gccgtaggtc agggtggtca cgagggtggg ccagggcacg ggcagcttgc
     2701 cggtggtgca gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc
     2761 cggacacgct gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca
     2821 ccccggtgaa cagctcctcg cccttgctca cctcgacgca taacttcgta taggatactt
     2881 tatacgaagt tatctttggg attcttccta ttttgatcca agcatcacca tcgaccctct
     2941 agtccagatc tcaccatcga cccataactt cgtatagcat acattatacg aagttatgtc
     3001 cctcgaagag gttcgaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa
     3061 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     3121 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     3181 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     3241 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     3301 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     3361 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     3421 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctatgttgcc acctggattc
     3481 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     3541 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     3601 ggatctccct ttgggccgcc tccccgcatc gataccgagc gctgctcgag agatctacgg
     3661 gtggcatccc tgtgacccct ccccagtgcc tctcctggcc ctggaagttg ccactccagt
     3721 gcccaccagc cttgtcctaa taaaattaag ttgcatcatt ttgtctgact aggtgtcctt
     3781 ctataatatt atggggtgga ggggggtggt atggagcaag gggcaagttg ggaagacaac
     3841 ctgtagggcc tgcggggtct attgggaacc aagctggagt gcagtggcac aatcttggct
     3901 cactgcaatc tccgcctcct gggttcaagc gattctcctg cctcagcctc ccgagttgtt
     3961 gggattccag gcatgcatga ccaggctcag ctaatttttg tttttttggt agagacgggg
     4021 tttcaccata ttggccaggc tggtctccaa ctcctaatct caggtgatct acccaccttg
     4081 gcctcccaaa ttgctgggat tacaggcgtg aaccactgct cccttccctg tccttctgat
     4141 tttgtaggta accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc
     4201 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     4261 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     4321 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     4381 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     4441 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     4501 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4561 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     4621 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     4681 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt
     4741 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     4801 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     4861 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     4921 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     4981 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     5041 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     5101 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     5161 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     5221 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     5281 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     5341 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     5401 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     5461 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     5521 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     5581 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     5641 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     5701 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     5761 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     5821 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     5881 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     5941 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     6001 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     6061 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     6121 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     6181 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     6241 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     6301 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     6361 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     6421 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     6481 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     6541 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     6601 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     6661 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     6721 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     6781 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     6841 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     6901 ttgctcacat gt
//
