LOCUS       sequence-12797-e        6323 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 6323)
  TITLE     Double Floxed Inverted ORF Control
REFERENCE   2  (bases 1 to 6323)
  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"
COMMENT     Sequence Label: pAAV-Ef1a-DIO EYFP
FEATURES             Location/Qualifiers
     source          1..6323
                     /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"
     protein_bind    complement(1450..1483)
                     /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(1534..1567)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(1576..2289,2290..2292,2293..2295))
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1621..1642)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1970..1989
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2229..2250
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      2292..2301
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2308..2341
                     /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    2392..2425
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2450..3038
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2503..2523)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2921..2932)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3070..3546
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3269..3288)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3586..3726
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3586..3715
                     /label=AAV2 ITR
     rep_origin      3801..4256
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3888..3907)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4098..4119
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4273..4292)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4392..4414
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4452..4470)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4538..4642
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4643..4711,4712..5503)
                     /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(4861..4880)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5674..6262
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6163..6182
                     /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 ctctagagtc
     1441 gactccggaa taacttcgta taggatactt tatacgaagt tatgcagaat ggtagctgga
     1501 ttgtagctgc tattagcaat atgaaacctc ttaataactt cgtatagcat acattatacg
     1561 aagttatggc gcgccttact tgtacagctc gtccatgccg agagtgatcc cggcggcggt
     1621 cacgaactcc agcaggacca tgtgatcgcg cttctcgttg gggtctttgc tcagggcgga
     1681 ctggtagctc aggtagtggt tgtcgggcag cagcacgggg ccgtcgccga tgggggtgtt
     1741 ctgctggtag tggtcggcga gctgcacgct gccgtcctcg atgttgtggc ggatcttgaa
     1801 gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta
     1861 gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag
     1921 ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt
     1981 gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt
     2041 gaagaagtcg tgctgcttca tgtggtcggg gtagcgggcg aagcactgca ggccgtagcc
     2101 gaaggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt
     2161 cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg
     2221 gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc
     2281 gcccttgctc accatggtgg cgctagcata acttcgtata aagtatccta tacgaagtta
     2341 tttgccttaa cccagaaatt atcactgtta ttctttagaa tggtgcaaag aataacttcg
     2401 tataatgtat gctatacgaa gttatgaatt cgatatcaag cttatcgata atcaacctct
     2461 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     2521 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     2581 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     2641 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     2701 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     2761 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     2821 caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct tggctgctcg cctatgttgc
     2881 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     2941 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     3001 tcagacgagt cggatctccc tttgggccgc ctccccgcat cgataccgag cgctgctcga
     3061 gagatctacg ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
     3121 gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac
     3181 taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
     3241 gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
     3301 caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct gcctcagcct
     3361 cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg
     3421 tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc
     3481 tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
     3541 gtccttctga ttttgtaggt aaccacgtgc ggaccgagcg gccgcaggaa cccctagtga
     3601 tggagttggc cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg
     3661 tcgcccgacg cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc
     3721 tgcaggggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     3781 tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt
     3841 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt
     3901 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct
     3961 ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg
     4021 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga
     4081 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc
     4141 gggctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga
     4201 gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg
     4261 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
     4321 aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
     4381 tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
     4441 gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
     4501 ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     4561 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     4621 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     4681 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     4741 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     4801 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     4861 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     4921 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     4981 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     5041 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     5101 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     5161 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     5221 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     5281 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     5341 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     5401 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     5461 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     5521 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     5581 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     5641 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     5701 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     5761 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     5821 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     5881 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     5941 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     6001 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     6061 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     6121 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     6181 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     6241 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     6301 ttgctggcct tttgctcaca tgt
//
