LOCUS       sequence-466637-        6348 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV cloning vector for CRE-dependent expression under the CAG 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6348)
  TITLE     Ritola AAV cloning vectors
REFERENCE   2  (bases 1 to 6348)
  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-FLEX-MCS-WPRE-SV40 - GCH2
FEATURES             Location/Qualifiers
     source          1..6348
                     /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    complement(1985..2018)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(2069..2102)
                     /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)."
     misc_feature    2103..2210
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     2119..2135
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2120..2136
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2170..2186)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2170..2186)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    2211..2244
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2295..2328
                     /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)."
     primer_bind     complement(2362..2381)
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     intron          2389..3405
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     complement(2443..2465)
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     promoter        complement(3406..3686)
                     /label=chicken beta-actin promoter
     enhancer        3688..4067
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   4097..4237
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   4097..4226
                     /label=AAV2 ITR
     primer_bind     complement(4247..4264)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4247..4263)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4256..4278)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4476..4931
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4563..4582)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4773..4794
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4948..4967)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5067..5089
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5127..5145)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5213..5317
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5318..5386,5387..6178)
                     /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(5536..5555)
                     /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 tagcataact tcgtatagca tacattatac gaagttattc tttgcaccat tctaaagaat
     2041 aacagtgata atttctgggt taaggcaaat aacttcgtat aggatacttt atacgaagtt
     2101 atggtaccgg gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc
     2161 agcccggggg atccactagt tctagagcgg ccgccaccgc ggtggagctc ataacttcgt
     2221 ataatgtatg ctatacgaag ttattaagag gtttcatatt gctaatagca gctacaatcc
     2281 agctaccatt ctgcataact tcgtataaag tatcctatac gaagttatac cggtcaattc
     2341 tttgccaaaa tgatgagaca gcacaataac cagcacgttg cccaggagct gtaggaaaaa
     2401 gaagaaggca tgaacatggt tagcagaggc tctagagccg ccggtcacac gccagaagcc
     2461 gaaccccgcc ctgccccgtc ccccccgaag gcagccgtcc ccccgcggac agccccgagg
     2521 ctggagaggg agaaggggac ggcggcgcgg cgacgcacga aggccctccc cgcccatttc
     2581 cttcctgccg gcgccgcacc gcttcgcccc gcgcccgcta gagggggtgc ggcggcgcct
     2641 cccagatttc ggctccgcac agatttggga caaaggaagt ccctgcgccc tctcgcacga
     2701 ttaccataaa aggcaatggc tgcggctcgc cgcgcctcga cagccgccgg cgctccgggg
     2761 gccgccgcgc ccctcccccg agccctcccc ggcccgaggc ggccccgccc cgcccggcac
     2821 ccccacctgc cgccaccccc cgcccggcac ggcgagcccc gcgccacgcc ccgtacggag
     2881 ccccgcaccc gaagccgggc cgtgctcagc aactcgggga ggggggtgca gggggggttg
     2941 cagcccgacc gacgcgccca caccccctgc tcaccccccc acgcacacac cccgcacgca
     3001 gcctttgttc ccctcgcagc cccccccgca ccgcggggca ccgcccccgg ccgcgctccc
     3061 ctcgcgcaca ctgcggagcg cacaaagccc cgcgccgcgc ccgcagcgct cacagccgcc
     3121 gggcagcgcg gagccgcacg cggcgctccc cacgcacaca cacacgcacg caccccccga
     3181 gccgctcccc ccgcacaaag ggccctcccg gagcccctca aggctttcac gcagccacag
     3241 aaaagaaaca agccgtcatt aaaccaagcg ctaattacag cccggaggag aagggccgtc
     3301 ccgcccgctc acctgtggga gtaacgcggt cagtcagagc cggggcgggc ggcgcgaggc
     3361 ggcggcggag cggggcacgg ggcgaaggca gcgcgcagcg actcccgccc gccgcgcgct
     3421 tcgcttttta tagggccgcc gccgccgccg cctcgccata aaaggaaact ttcggagcgc
     3481 gccgctctga ttggctgccg ccgcacctct ccgcctcgcc ccgccccgcc cctcgccccg
     3541 ccccgccccg cctggcgcgc gccccccccc cccccccccc gcccccatcg ctgcacaaaa
     3601 taattaaaaa ataaataaat acaaaattgg gggtggggag gggggggaga tggggagagt
     3661 gaagcagaac gtggggctca cctcgaccat ggtaatagcg atgactaata cgtagatgta
     3721 ctgccaagta ggaaagtccc ataaggtcat gtactgggca taatgccagg cgggccattt
     3781 accgtcattg acgtcaatag ggggcgtact tggcatatga tacacttgat gtactgccaa
     3841 gtgggcagtt taccgtaaat actccaccca ttgacgtcaa tggaaagtcc ctattggcgt
     3901 tactatggga acatacgtca ttattgacgt caatgggcgg gggtcgttgg gcggtcagcc
     3961 aggcgggcca tttaccgtaa gttatgtaac gcggaactcc atatatgggc tatgaactaa
     4021 tgaccccgta attgattact attaataact agtcaataat caatgtcgag ggttaccgcc
     4081 taggatcgat agatctagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     4141 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     4201 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggcag cttggcactg gccgtcgttt
     4261 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     4321 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     4381 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg
     4441 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
     4501 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     4561 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     4621 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     4681 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     4741 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     4801 actcaaccct atctcgggct attcttttga tttataaggg attttgccga tttcggccta
     4861 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     4921 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
     4981 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5041 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5101 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
     5161 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
     5221 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     5281 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     5341 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     5401 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     5461 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     5521 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     5581 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     5641 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     5701 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     5761 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     5821 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     5881 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     5941 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     6001 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     6061 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     6121 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     6181 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     6241 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     6301 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttc
//
