LOCUS       sequence-135058-        3300 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  gap-repair recombineering for Drosophila transgenesis, contains loxP
            cassette with GFP, chloramphenicol resistant.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3300)
  AUTHORS   Haussmann IU, Ustaoglu P, Brauer U, Hemani Y, Dix TC, Soller M
  TITLE     Plasmid-based gap-repair recombineered transgenes reveal a central 
            role for introns in mutually exclusive alternative splicing in Down 
            Syndrome Cell Adhesion Molecule exon 4.
  JOURNAL   Nucleic Acids Res. 2018 Dec 12. pii: 5239037. doi: 
            10.1093/nar/gky1254.
  PUBMED    30541104
REFERENCE   2  (bases 1 to 3300)
  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: "Nucleic 
            Acids Res. 2018 Dec 12. pii: 5239037. doi: 10.1093/nar/gky1254."
COMMENT     Sequence Label: pOT2 3GLA
FEATURES             Location/Qualifiers
     source          1..3300
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    188..257
                     /label=attB
                     /bound_moiety="phage phi-C31 integrase"
                     /note="attB site for the phi-C31 integrase (Groth et al., 
                     2000)"
     primer_bind     complement(358..377)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        complement(385..403)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(386..403)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             complement(468..1127)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1042..1061
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1164..1169,1170..1186,1187..1192))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      complement(1254..1842)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1334..1353)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1946..1965
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1946..1964
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2108..2141
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     regulatory      2377..2386
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2398..3108
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     CDS             2398..3108
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKF
                     ICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN
                     YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVN
                     FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEF
                     VTAAGITLGMDELYK"
     primer_bind     complement(2437..2458)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2698..2717)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3045..3066
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    3255..3288
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
ORIGIN
        1 tgcccgccgt gaccgtcgag aacccgctga cgctgccccg cgtatccgca cccgccgacg
       61 ccgtcgcacg tcccgtgctc accgtgacca ccgcgcccag cggtttcgag ggcgagggct
      121 tcccggtgcg ccgcgcgttc gccgggatca actaccgcca cctcgacccg ttcatcatga
      181 tggaccagat gggtgaggtg gagtacgcgc ccggggagcc caagggcacg ccctggcacc
      241 cgcaccgcgg cttcgagacc tgacctacat cgtaccggct tgacgacctt ggagcgactg
      301 tcgttagttc cgcgcgattc ggtgcggtat ttcacaccgc atatggtgca ctctcagtac
      361 aatctgctct gatgccgctc gagttctata gtgtcaccta aatcgtatgt gtatgataca
      421 taaggttatg tattaattgt agccgcgttc taacgtcgac gtaccaatta cgccccgccc
      481 tgccactcat cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca
      541 caaacggcat gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa
      601 tatttgccca tggtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca
      661 aaactggtga aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct
      721 ttagggaaat aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga
      781 aactgccgga aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca
      841 tggaaaacgg tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt
      901 gccatacgga actccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga
      961 taaaacttgt gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg
     1021 gtctggttat aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc
     1081 cattgggata tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta
     1141 gctcctgaaa atctcgataa ctcattaaag cttatcgatg ataagctgtc aaacatgaga
     1201 atttagcccg cctaatgagc gggctttttg ttgtgatccg cgttgctggc gtttttccat
     1261 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
     1321 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
     1381 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg
     1441 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg
     1501 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt
     1561 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg
     1621 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac
     1681 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga
     1741 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt
     1801 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttggatcac
     1861 aaaaaaaagc ccgctcatta ggcgggttaa attcccgggt actgcagccg attcattaat
     1921 gcaggttaac ctggcttatc gaaattaata cgactcacta tagggagacc ggcagatctg
     1981 atatcatcgc cactgtgctg ggaattctgt cgacagctgc agtactagtc aagcttagga
     2041 tccgatatcc atggtgctag cgagatcttc ctcgagggta ccggcggccg cgtgcggccg
     2101 cgtctagata acttcgtata atgtatgcta tacgaagtta tcatgccggg gatctaattc
     2161 aattagagac taattcaatt agagctaatt caattaggat ccaagcttat cgatttcgaa
     2221 ccctcgaccg ccggagtata aatagaggcg cttcgtctac ggagcgacaa ttcaattcaa
     2281 acaagcaaag tgaacacgtc gctaagcgaa agctaagcaa ataaacaagc gcagttgaac
     2341 aagctaaaca atcgggcggc cgtactagag ccggtcgcca ccatggtgtc gagcgcaagc
     2401 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2461 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     2521 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     2581 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     2641 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2701 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2761 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2821 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     2881 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2941 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3001 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3061 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta ctcagatctt
     3121 gcaagcttgt agagtttccc atttaataat tcatattatc tcgaatctag tcaattacgg
     3181 ctttcctcaa atagaaaaat aaaaaaaatg aaaaaatgca cttgccattt aaacttagac
     3241 gcgataagct tgatataact tcgtataatg tatgctatac gaagttatac gcgtactaga
//
