LOCUS       sequence-481119-        5783 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV cloning vector for Flp dependent expression under the EF1a 
            promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5783)
  TITLE     Ritola Lab plasmids
REFERENCE   2  (bases 1 to 5783)
  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: EF1a-FRT-MCSrev-WPRE-SV40 - GCH18.1
FEATURES             Location/Qualifiers
     source          1..5783
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   1007..1147
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   1018..1147
                     /label=AAV2 ITR
     polyA_signal    1253..1374
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1290..1309)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1344..1363
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    1390..1978
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1496..1507
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1905..1925
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    1999..2032
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     protein_bind    2061..2108
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     misc_feature    2109..2216
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     2133..2149
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2133..2149
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(2183..2199)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2184..2200)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     protein_bind    complement(2217..2250)
                     /label=FRT3
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision with other FRT3 sites
                     (Bosch et al., 2015)"
     protein_bind    complement(2293..2340)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     promoter        complement(2347..3525)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2356..3294
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2379..2399)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     repeat_region   3532..3672
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3532..3661
                     /label=AAV2 ITR
     primer_bind     complement(3682..3699)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3682..3698)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3691..3713)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3911..4366
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3998..4017)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4208..4229
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4383..4402)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4502..4524
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4562..4580)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4648..4752
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4753..4821,4822..5613)
                     /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(4971..4990)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gaattgcctg caggcagctg
     1021 cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt
     1081 cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg
     1141 ggttcctatc gatggcgcgc ctaccacatt tgtagaggtt ttacttgctt taaaaaacct
     1201 cccacatctc cccctgaacc tgaaacataa aatgaatgca attgttgttg ttaacttgtt
     1261 tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
     1321 atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt
     1381 ctgtgtacag cggggaggcg gcccaaaggg agatccgact cgtctgaggg cgaaggcgaa
     1441 gacgcggaag aggccgcaga gccggcagca ggccgcggga aggaaggtcc gctggattga
     1501 gggccgaagg gacgtagcag aaggacgtcc cgcgcagaat ccaggtggca acacaggcga
     1561 gcagccaagg aaaggacgat gatttccccg acaacaccac ggaattgtca gtgcccaaca
     1621 gccgagcccc tgtccagcag cgggcaaggc aggcggcgat gagttccgcc gtggcaatag
     1681 ggagggggaa agcgaaagtc ccggaaagga gctgacaggt ggtggcaatg ccccaaccag
     1741 tgggggttgc gtcagcaaac acagtgcaca ccacgccacg ttgcctgaca acgggccaca
     1801 actcctcata aagagacagc aaccaggatt tatacaagga ggagaaaatg aaagccatac
     1861 gggaagcaat agcatgatac aaaggcatta aagcagcgta tccacatagc gtaaaaggag
     1921 caacatagtt aagaatacca gtcaatcttt cacaaatttt gtaatccaga ggttgattgc
     1981 tagcgaagtt cctattccga agttcctatt cttcaaatag tataggaact tcttggatca
     2041 aaataggagc gatcgcaatg gaagttccta ttccgaagtt cctattctct agaaagtata
     2101 ggaacttcga gctccaccgc ggtggcggcc gctctagaac tagtggatcc cccgggctgc
     2161 aggaattcga tatcaagctt atcgataccg tcgacctcga gggggggccc ggtaccgaag
     2221 ttcctatact atttgaagaa taggaacttc ggaataggaa cttcagagcg ctttggccgc
     2281 tccggccaaa gggaagttcc tatactttct agagaatagg aacttcggaa taggaacttc
     2341 accggttcac gacacctgaa atggaagaaa aaaactttga accactgtct gaggcttgag
     2401 aatgaaccaa gatccaaact caaaaagggc aaattccaag gagaattaca tcaagtgcca
     2461 agctggccta acttcagtct ccacccactc agtgtgggga aactccatcg cataaaaccc
     2521 ctccccccaa cctaaagacg acgtactcca aaagctcgag aactaatcga ggtgcctgga
     2581 cggcgcccgg tactccgtgg agtcacatga agcgacggct gaggacggaa aggccctttt
     2641 cctttgtgtg ggtgactcac ccgcccgctc tcccgagcgc cgcgtcctcc attttgagct
     2701 ccctgcagca gggccgggaa gcggccatct ttccgctcac gcaactggtg ccgaccgggc
     2761 cagccttgcc gcccagggcg gggcgataca cggcggcgcg aggccaggca ccagagcagg
     2821 ccggccagct tgagactacc cccgtccgat tctcggtggc cgcgctcgca ggccccgcct
     2881 cgccgaacat gtgcgctggg acgcacgggc cccgtcgccg cccgcggccc caaaaaccga
     2941 aataccagtg tgcagatctt ggcccgcatt tacaagacta tcttgccaga aaaaaagcgt
     3001 cgcagcaggt catcaaaaat tttaaatggc tagagactta tcgaaagcag cgagacaggc
     3061 gcgaaggtgc caccagattc gcacgcggcg gccccagcgc ccaggccagg cctcaactca
     3121 agcacgaggc gaaggggctc cttaagcgca aggcctcgaa ctctcccacc cacttccaac
     3181 ccgaagctcg ggatcaagaa tcacgtactg cagccaggtg gaagtaattc aaggcacgca
     3241 agggccataa cccgtaaaga ggccaggccc gcgggaacca cacacggcac ttacctgtgt
     3301 tctggcggca aacccgttgc gaaaaagaac gttcacggcg actactgcac ttatatacgg
     3361 ttctccccca ccctcgggaa aaaggcggag ccagtacacg acatcacttt cccagtttac
     3421 cccgcgccac cttctctagg caccggttca attgccgacc cctcccccca acttctcggg
     3481 gactgtgggc gatgtgcgct ctgcccactg acgggcaccg gagccagatc taggaacccc
     3541 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac
     3601 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca
     3661 gctgcctgca ggcagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc
     3721 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag
     3781 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg
     3841 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag
     3901 caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     3961 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     4021 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     4081 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca
     4141 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     4201 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc gggctattct
     4261 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     4321 caaaaattta acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc
     4381 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     4441 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     4501 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag
     4561 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     4621 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4681 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4741 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4801 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4861 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4921 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4981 gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     5041 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     5101 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5161 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5221 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5281 gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5341 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5401 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5461 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5521 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5581 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5641 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5701 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5761 gtagaaaaga tcaaaggatc ttc
//
