LOCUS       sequence-434052-        3769 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  insert vector for Switch-OVER: Single loxP-STOP-EF1a-Blasti-mU6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3769)
  AUTHORS   Chylinski K, Hubmann M, Hanna RE, Yanchus C, Michlits G, Uijttewaal 
            ECH, Doench J, Schramek D, Elling U
  TITLE     CRISPR-Switch regulates sgRNA activity by Cre recombination for 
            sequential editing of two loci.
  JOURNAL   Nat Commun. 2019 Nov 29;10(1):5454. doi: 10.1038/s41467-019-13403-y.
  PUBMED    31784531
REFERENCE   2  (bases 1 to 3769)
  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: "Nat 
            Commun."; date: "2019-11-29"; volume: "10(1):5454. doi"; pages: " 
            10.1038/s41467-019-13403-y"
COMMENT     Sequence Label: p931-SWITCH-OVER insert: EF1a-Blasti
FEATURES             Location/Qualifiers
     source          1..3769
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(99..167,168..959)
                     /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(317..336)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1130..1718
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1619..1638
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1872..1889
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2006..2027
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2042..2047,2048..2065,2066..2072)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2080..2096
                     /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     2085..2107
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2104..2120
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2104..2120
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(2179..2212)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2278..2489
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2508..2517
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2514..2912
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     promoter        2913..3226
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for mouse U6 snRNA (Das 
                     et al., 1988)"
     primer_bind     3145..3165
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     primer_bind     complement(3273..3290)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3273..3289)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3282..3304)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(3498..3517)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3617..3639
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3677..3695)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(3763..3769,1..98)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
       61 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
      121 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
      181 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
      241 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
      301 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
      361 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
      421 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
      481 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
      541 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
      601 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
      661 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
      721 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
      781 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
      841 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
      901 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
      961 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     1021 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     1081 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1141 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1201 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1261 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     1321 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     1381 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     1441 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     1501 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     1561 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     1621 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     1681 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     1741 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     1801 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     1861 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     1921 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     1981 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     2041 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     2101 acacaggaaa cagctatgac catgattacg ccaagcttgc atgcctgcag gtcgaagacc
     2161 tgtttaagag ctatgctgat aacttcgtat agcatacatt atacgaagtt attttttttt
     2221 tttgcggccg cccagaattg acgcgtggag tgggaattgg ctccggtgcc cgtcagtggg
     2281 cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc aattgatccg
     2341 gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac tggctccgcc
     2401 tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg aacgttcttt
     2461 ttcgcaacgg gtttgccgcc agaacacagg accggttcta gagcgctgcc accatggcca
     2521 agcctttgtc tcaagaagaa tccaccctca ttgaaagagc aacggctaca atcaacagca
     2581 tccccatctc tgaggattac agcgtcgcca gcgcagctct ctctagcgac ggccgcatct
     2641 tcactggtgt caatgtatat cattttactg ggggaccttg tgcagaactc gtggtgctgg
     2701 gcactgctgc tgctgcggca gctggcaacc tgacttgtat cgtcgcgatc ggaaatgaga
     2761 acaggggcat cttgagcccc tgcggacggt gccgacaggt gcttctcgat ctgcatcctg
     2821 ggatcaaagc catagtgaag gacagtgatg gacagccgac ggcagttggg attcgtgaat
     2881 tgctgccctc tggttatgtg tgggagggct gagatccgac gccgccatct ctaggcccgc
     2941 gccggccccc tcgcacagac ttgtgggaga agctcggcta ctcccctgcc ccggttaatt
     3001 tgcatataat atttcctagt aactatagag gcttaatgtg cgataaaaga cagataatct
     3061 gttcttttta atactagcta cattttacat gataggcttg gatttctata agagatacaa
     3121 atactaaatt attattttaa aaaacagcac aaaaggaaac tcaccctaac tgtaaagtaa
     3181 ttgtgtgttt tgagactata aatatccctt ggagaaaagc cttgtttggg gtcttcgact
     3241 ctagaggatc cccgggtacc gagctcgaat tcactggccg tcgttttaca acgtcgtgac
     3301 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     3361 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     3421 ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     3481 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     3541 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3601 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3661 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     3721 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaa
//
