LOCUS       sequence-307589-        6646 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cre-dependent expression of Flp recombinase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6646)
  AUTHORS   Fischer KB, Collins HK, Callaway EM
  TITLE     Sources of off-target expression from recombinase-dependent AAV 
            vectors and mitigation with cross-over insensitive ATG-out vectors.
  JOURNAL   Proc Natl Acad Sci U S A. 2019 Dec 16. pii: 1915974116. doi: 
            10.1073/pnas.1915974116.
  PUBMED    31843925
REFERENCE   2  (bases 1 to 6646)
  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"; journalName: "Proc Natl 
            Acad Sci U S A. 2019 Dec 16. pii: 1915974116. doi: 
            10.1073/pnas.1915974116."
COMMENT     Sequence Label: pAAV-nef-CIAO-Flp
FEATURES             Location/Qualifiers
     source          1..6646
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        177..388
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             401..669
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     regulatory      700..709
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    complement(711..744)
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     intron          1265..1330
                     /label=small t intron
                     /note="SV40 (simian virus 40) small t antigen intron"
     polyA_signal    1908..2042
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1945..1964)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1999..2018
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(2062..2095)
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    3393..3869
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(3592..3611)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     repeat_region   3909..4049
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3909..4038
                     /label=AAV2 ITR
     rep_origin      4124..4579
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4211..4230)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4421..4442
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4596..4615)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4715..4737
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4775..4793)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4861..4965
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4966..5034,5035..5826)
                     /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(5184..5203)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5997..6585
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6486..6505
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct gcggccgcac gcgtaaggat ctgcgatcgc tccggtgccc gtcagtgggc
      181 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgaacggg
      241 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct
      301 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt
      361 tcgcaacggg tttgccgcca gaacacagct gaagcttcga ggggctcgca tctctccttc
      421 acgcgcccgc cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc
      481 tcccgcctgt ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa agctcaggtc
      541 gagaccgggc ctttgtccgg cgctcccttg gagcctacct agactcagcc ggctctccac
      601 gctttgcctg accctgcttg ctcaactcta cgtctttgtt tcgttttctg ttctgcgccg
      661 ttacagatcc aagctgtgac cggcgcctac ggatccgccg ccaccatggg taccgttcgt
      721 atagcataca ttatacgaag ttatgagctc tcggggacac caaatatggc gatctcggcc
      781 ttttcgtttc ttggagctgg gacatgtttg ccatcgatcc atctaccacc agaacggccg
      841 ttagatctgc tgccaccgtt gtttccaccg aagaaaccac cgttgccgta accaccacga
      901 cggttgttgc taaagaagct gccaccgcca cggccaccgt tgtagccgcc gttgttgtta
      961 ttgtagttgc tactgttatt tctggcactt cttggttttc ctcttaagtg aggaggaaca
     1021 taaccattct cgttgttgtc gttgatgctt aaattttgca cttgttcgct cagttcagcc
     1081 ataatatgaa atgcttttct tttgttctta cggaatacca cttgccacct atcaccacaa
     1141 ctaacttttt cccgttcctc catctctttt atattttttt tctcgaggga tctttgtgaa
     1201 ggaaccttac ttctgtggtg tgacataatt ggacaaacta cctacagaga tttaaagctc
     1261 taaggtaaat ataaaatttt taagtgtata atgtgttaaa ctactgattc taattgtttg
     1321 tgtattttag attccaacct atggaactga tgaatgggag cagtggtgga atgcctttaa
     1381 tgaggaaaac ctgttttgct cagaagaaat gccatctagt gatgatgagg ctactgctga
     1441 ctctcaacat tctactcctc caaaaaagaa gaagagaaag gtagaagacc ccaaggactt
     1501 tccttcagaa ttgctaagtt ttttgagtca tgctgtgttt agtaatagaa ctcttgcttg
     1561 ctttgctatt tacaccacaa aggaaaaagc tgcactgcta tacaagaaaa ttatggaaaa
     1621 atattctgta acctttataa gtaggcataa cagttataat cataacatac tgttttttct
     1681 tactccacac aggcatagag tgtctgctat taataactat gctcaaaaat tgtgtacctt
     1741 tagcttttta atttgtaaag gggttaataa ggaatatttg atgtatagtg ccttgactag
     1801 agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     1861 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     1921 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     1981 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     2041 tctgacatgg taagtgaatt cataacttcg tatagcatac attatacgaa cggtagctag
     2101 cagccagttc gacatcctgt gcaagacccc ccccaaggtg ctggtgcggc agttcgtgga
     2161 gagattcgag aggcccagcg gcgagaagat cgccagctgt gccgccgagc tgacctacct
     2221 gtgctggatg atcacccaca acggcaccgc catcaagagg gccaccttca tgagctacaa
     2281 caccatcatc agcaacagcc tgagcttcga catcgtgaac aagagcctgc agttcaagta
     2341 caagacccag aaggccacca tcctggaggc cagcctgaag aagctgatcc ccgcctggga
     2401 gttcaccatc atcccttaca acggccagaa gcaccagagc gacatcaccg acatcgtgtc
     2461 cagcctgcag ctgcagttcg agagcagcga ggaggccgac aagggcaaca gccacagcaa
     2521 gaagatgctg aaggccctgc tgtccgaggg cgagagcatc tgggagatca ccgagaagat
     2581 cctgaacagc ttcgagtaca ccagcaggtt caccaagacc aagaccctgt accagttcct
     2641 gttcctggcc acattcatca actgcggcag gttcagcgac atcaagaacg tggaccccaa
     2701 gagcttcaag ctggtgcaga acaagtacct gggcgtgatc attcagtgcc tggtgaccga
     2761 gaccaagaca agcgtgtcca ggcacatcta ctttttcagc gccagaggca ggatcgaccc
     2821 cctggtgtac ctggacgagt tcctgaggaa cagcgagccc gtgctgaaga gagtgaacag
     2881 gaccggcaac agcagcagca acaagcagga gtaccagctg ctgaaggaca acctggtgcg
     2941 cagctacaac aaggccctga agaagaacgc cccctacccc atcttcgcta tcaagaacgg
     3001 ccctaagagc cacatcggca ggcacctgat gaccagcttt ctgagcatga agggcctgac
     3061 cgagctgaca aacgtggtgg gcaactggag cgacaagagg gcctccgccg tggccaggac
     3121 cacctacacc caccagatca ccgccatccc cgaccactac ttcgccctgg tgtccaggta
     3181 ctacgcctac gaccccatca gcaaggagat gatcgccctg aaggacgaga ccaaccccat
     3241 cgaggagtgg cagcacatcg agcagctgaa gggcagcgcc gagggcagca tcagataccc
     3301 cgcctggaac ggcatcatca gccaggaggt gctggactac ctgagcagct acatcaacag
     3361 gcggatctga ggcgcgccct cgagagatct acgggtggca tccctgtgac ccctccccag
     3421 tgcctctcct ggccctggaa gttgccactc cagtgcccac cagccttgtc ctaataaaat
     3481 taagttgcat cattttgtct gactaggtgt ccttctataa tattatgggg tggagggggg
     3541 tggtatggag caaggggcaa gttgggaaga caacctgtag ggcctgcggg gtctattggg
     3601 aaccaagctg gagtgcagtg gcacaatctt ggctcactgc aatctccgcc tcctgggttc
     3661 aagcgattct cctgcctcag cctcccgagt tgttgggatt ccaggcatgc atgaccaggc
     3721 tcagctaatt tttgtttttt tggtagagac ggggtttcac catattggcc aggctggtct
     3781 ccaactccta atctcaggtg atctacccac cttggcctcc caaattgctg ggattacagg
     3841 cgtgaaccac tgctcccttc cctgtccttc tgattttgta ggtaaccacg tgcggaccga
     3901 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     3961 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     4021 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     4081 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     4141 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4201 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4261 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4321 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     4381 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     4441 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     4501 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     4561 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     4621 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     4681 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     4741 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     4801 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     4861 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     4921 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     4981 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     5041 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     5101 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     5161 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     5221 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     5281 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     5341 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     5401 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     5461 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     5521 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     5581 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     5641 ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca
     5701 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     5761 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     5821 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     5881 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     5941 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     6001 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     6061 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     6121 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     6181 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     6241 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     6301 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     6361 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     6421 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     6481 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     6541 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     6601 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
