LOCUS       sequence-487174-        6357 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV cloning vector for Flp dependent expression under the CAG 
            promoter. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6357)
  TITLE     Ritola Lab plasmids
REFERENCE   2  (bases 1 to 6357)
  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: CAG-FRT-MCSrev-WPRE-SV40 - GCH6.1
FEATURES             Location/Qualifiers
     source          1..6357
                     /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)."
     primer_bind     complement(2374..2393)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          2401..3417
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(2455..2477)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     enhancer        3697..4076
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   4106..4246
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4106..4235
                     /label=AAV2 ITR
     primer_bind     complement(4256..4273)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4256..4272)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4265..4287)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4485..4940
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4572..4591)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4782..4803
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4957..4976)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5076..5098
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5136..5154)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5222..5326
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5327..5395,5396..6187)
                     /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(5545..5564)
                     /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 accggtcaat tctttgccaa aatgatgaga cagcacaata accagcacgt tgcccaggag
     2401 ctgtaggaaa aagaagaagg catgaacatg gttagcagag gctctagagc cgccggtcac
     2461 acgccagaag ccgaaccccg ccctgccccg tcccccccga aggcagccgt ccccccgcgg
     2521 acagccccga ggctggagag ggagaagggg acggcggcgc ggcgacgcac gaaggccctc
     2581 cccgcccatt tccttcctgc cggcgccgca ccgcttcgcc ccgcgcccgc tagagggggt
     2641 gcggcggcgc ctcccagatt tcggctccgc acagatttgg gacaaaggaa gtccctgcgc
     2701 cctctcgcac gattaccata aaaggcaatg gctgcggctc gccgcgcctc gacagccgcc
     2761 ggcgctccgg gggccgccgc gccccycccc cgagcccycc ccggcccgmg gcggccccgc
     2821 cccgcccggc acccccmcct gccgccaccc cccgcccggc acggcgagcc ccgcgccacg
     2881 ccccgtacgg agccccgcac ccgaagccgg gccgtgctca gcaactcggg gaggggggtg
     2941 cagggggggt tgcagcccga ccgacgcgcc cacaccccct gctcaccccc ccacgcacac
     3001 accccgcacg cagcctttgt tcccctcgca gccccccccg caccgcgggg caccgccccc
     3061 ggccgcgctc ccctcgcgca cactgcggag cgcacaaagc cccgcgccgc gcccgcagcg
     3121 ctcacagccg ccgggcagcg cggagccgca cgcggcgctc cccacgcaca cacacacgca
     3181 cgcacccccc gagccgctcc ccccgcacaa agggccctcc cggagcccct caaggctttc
     3241 acgcagccac agaaaagaaa caagccgtca ttaaaccaag cgctaattac agcccggagg
     3301 agaagggccg tcccgcccgc tcacctgtgg gagtaacgcg gtcagtcaga gccggggcgg
     3361 gcggcgcgag gcggcggcgg agcggggcac ggggcgaagg cagcgcgcag cgactcccgc
     3421 ccgccgcgcg cttcgctttt tatagggccg ccgccgccgc cgcctcgcca taaaaggaaa
     3481 ctttcggagc gcgccgctct gattggctgc cgccgcmccy cyccsccycs ccccsccccs
     3541 ccccccsccc cccccccccc cccccggcgc gcgccccccc cccccccccg cccccatcgc
     3601 tgcacaaaat aattaaaaaa taaataaata caaaattggg ggtggggagg ggggggagat
     3661 ggggagagtg aagcagaacg tggggctcac ctcgaccatg gtaatagcga tgactaatac
     3721 gtagatgtac tgccaagtag gaaagtccca taaggtcatg tactgggcat aatgccaggc
     3781 gggccattta ccgtcattga cgtcaatagg gggcgtactt ggcatatgat acacttgatg
     3841 tactgccaag tgggcagttt accgtaaata ctccacccat tgacgtcaat ggaaagtccc
     3901 tattggcgtt actatgggaa catacgtcat tattgacgtc aatgggcggg ggtcgttggg
     3961 cggtcagcca ggcgggccat ttaccgtaag ttatgtaacg cggaactcca tatatgggct
     4021 atgaactaat gaccccgtaa ttgattacta ttaataacta gtcaataatc aatgtcgagg
     4081 gttaccgcct aggatcgata gatctaggaa cccctagtga tggagttggc cactccctct
     4141 ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg cccgggcttt
     4201 gcccgggcgg cctcagtgag cgagcgagcg cgcagctgcc tgcaggcagc ttggcactgg
     4261 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
     4321 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
     4381 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
     4441 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
     4501 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     4561 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     4621 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     4681 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     4741 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     4801 tggaacaaca ctcaacccta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     4861 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4921 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     4981 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     5041 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     5101 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     5161 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     5221 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     5281 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     5341 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     5401 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     5461 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     5521 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     5581 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     5641 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     5701 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     5761 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     5821 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     5881 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     5941 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     6001 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     6061 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     6121 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     6181 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     6241 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     6301 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttc
//
