LOCUS       sequence-99405-e        3541 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR-RMCE, second step attB plasmid, intron phase 2, exchange 
            dsRed with 2xTY1 and V5 tags cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3541)
  AUTHORS   Zhang X, Koolhaas WH, Schnorrer F
  TITLE     A Versatile Two-Step CRISPR- and RMCE-Based Strategy for Efficient 
            Genome Engineering in Drosophila.
  JOURNAL   G3 (Bethesda). 2014 Oct 15. pii: g3.114.013979. doi: 
            10.1534/g3.114.013979.
  PUBMED    25324299
REFERENCE   2  (bases 1 to 3541)
  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: "G3 
            (Bethesda). 2014 Oct 15. pii: g3.114.013979. doi: 
            10.1534/g3.114.013979."
COMMENT     Sequence Label: pBS-KS-attB1-2-PT-SA-SD-2-2xTY1-V5
FEATURES             Location/Qualifiers
     source          1..3541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(83..100)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(254..842)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(334..353)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1013..1804,1805..1873))
                     /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     1636..1655
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1874..1978)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2004..2459)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2141..2162)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2353..2372
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2586..2608
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2600..2617
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2601..2617
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2627..2646
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2627..2645
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2681..2750
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     CDS             3017..3058
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     protein_bind    3245..3314
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     primer_bind     complement(3354..3374)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3354..3372)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3393..3409)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3393..3409)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3406..3428)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3417..3433
                     /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)."
     promoter        complement(join(3441..3447,3448..3465,3466..3471))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3486..3507
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
       61 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
      121 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
      181 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
      241 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      301 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      361 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      421 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
      481 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
      541 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
      601 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      661 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
      721 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      781 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
      841 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
      901 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
      961 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     1021 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     1081 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     1141 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     1201 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     1261 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     1321 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     1381 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     1441 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     1501 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     1561 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     1621 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     1681 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     1741 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     1801 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     1861 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     1921 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     1981 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt
     2041 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta
     2101 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc
     2161 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg
     2221 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact
     2281 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt
     2341 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc
     2401 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc
     2461 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
     2521 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
     2581 gttttcccag tcacgacgtt gtaaaacgac ggccagtgag cgcgcgtaat acgactcact
     2641 atagggcgaa ttggagctcg ctagcgatgt aggtcacggt ctcgaagccg cggtgcgggt
     2701 gccagggcgt gcccttgggc tccccgggcg cgtactccac ctcacccatc tggtccatca
     2761 tgatggtcga ctctagaagt cgatccaaca tggcgacttg tcccatcccc ggcatgttta
     2821 aatatactaa ttattcttga actaatttta atcaaccgat ttatctctct tccgcaggtc
     2881 gggaggttcc ggtggaagcg gaggtagcgg cggatcggag gtccacacta atcaagaccc
     2941 cctggatgcc gaggtgcaca ccaaccagga ccctctggac ggaggttccg gtggaagcgg
     3001 aggtagcggc ggatcgggca agcccatccc caaccccctg ctgggcctgg atagcaccag
     3061 atccggaggt agcggtggaa gcggaggttc cggcaggtaa gttattgaac aatggcatca
     3121 aatgccttca tcatcactac cctttagccc ttaagacccc acaatgacct tacccactca
     3181 gagaaaaaag taaatatgaa agcccatttg aacttctaag cttgtcgacc atcatgatgg
     3241 accagatggg tgaggtggag tacgcgcccg gggagcccaa gggcacgccc tggcacccgc
     3301 accgcggctt cgagaccgtg acctacatca tgcatggtac ccagcttttg ttccctttag
     3361 tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3421 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3481 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3541 g
//
