LOCUS       sequence-457139-        5771 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AAV cloning vector for CRE-dependent expression under the EF1a 
            promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5771)
  TITLE     Ritola AAV cloning vectors
REFERENCE   2  (bases 1 to 5771)
  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-Flex-MCS-WPRE-SV40 - GCH14
FEATURES             Location/Qualifiers
     source          1..5771
                     /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)."
     promoter        complement(2335..3513)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2344..3282
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(2367..2387)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     repeat_region   3520..3660
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3520..3649
                     /label=AAV2 ITR
     primer_bind     complement(3670..3687)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3670..3686)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3679..3701)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3899..4354
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3986..4005)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4196..4217
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4371..4390)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4490..4512
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4550..4568)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4636..4740
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4741..4809,4810..5601)
                     /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(4959..4978)
                     /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 cggttcacga
     2341 cacctgaaat ggaagaaaaa aactttgaac cactgtctga ggcttgagaa tgaaccaaga
     2401 tccaaactca aaaagggcaa attccaagga gaattacatc aagtgccaag ctggcctaac
     2461 ttcagtctcc acccactcag tgtggggaaa ctccatcgca taaaacccct ccccccaacc
     2521 taaagacgac gtactccaaa agctcgagaa ctaatcgagg tgcctggacg gcgcccggta
     2581 ctccgtggag tcacatgaag cgacggctga ggacggaaag gcccttttcc tttgtgtggg
     2641 tgactcaccc gcccgctctc ccgagcgccg cgtcctccat tttgagctcc ctgcagcagg
     2701 gccgggaagc ggccatcttt ccgctcacgc aactggtgcc gaccgggcca gccttgccgc
     2761 ccagggcggg gcgatacacg gcggcgcgag gccaggcacc agagcaggcc ggccagcttg
     2821 agactacccc cgtccgattc tcggtggccg cgctcgcagg ccccgcctcg ccgaacatgt
     2881 gcgctgggac gcacgggccc cgtcgccgcc cgcggcccca aaaaccgaaa taccagtgtg
     2941 cagatcttgg cccgcattta caagactatc ttgccagaaa aaaagcgtcg cagcaggtca
     3001 tcaaaaattt taaatggcta gagacttatc gaaagcagcg agacaggcgc gaaggtgcca
     3061 ccagattcgc acgcggcggc cccagcgccc aggccaggcc tcaactcaag cacgaggcga
     3121 aggggctcct taagcgcaag gcctcgaact ctcccaccca cttccaaccc gaagctcggg
     3181 atcaagaatc acgtactgca gccaggtgga agtaattcaa ggcacgcaag ggccataacc
     3241 cgtaaagagg ccaggcccgc gggaaccaca cacggcactt acctgtgttc tggcggcaaa
     3301 cccgttgcga aaaagaacgt tcacggcgac tactgcactt atatacggtt ctcccccacc
     3361 ctcgggaaaa aggcggagcc agtacacgac atcactttcc cagtttaccc cgcgccacct
     3421 tctctaggca ccggttcaat tgccgacccc tccccccaac ttctcgggga ctgtgggcga
     3481 tgtgcgctct gcccactgac gggcaccgga gccagatcta ggaaccccta gtgatggagt
     3541 tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc
     3601 gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc tgcctgcagg
     3661 cagcttggca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca
     3721 acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg
     3781 caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta
     3841 ttttctcctt acgcatctgt gcggtatttc acaccgcata cgtcaaagca accatagtac
     3901 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3961 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     4021 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     4081 gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg tagtgggcca
     4141 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4201 ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt tgatttataa
     4261 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4321 gcgaatttta acaaaatatt aacgtttaca attttatggt gcactctcag tacaatctgc
     4381 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga
     4441 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4501 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata
     4561 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     4621 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     4681 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     4741 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     4801 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4861 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4921 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     4981 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     5041 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     5101 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     5161 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     5221 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     5281 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     5341 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     5401 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     5461 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     5521 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     5581 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     5641 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     5701 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     5761 aaaggatctt c
//
