LOCUS       sequence-9308-ex        6051 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6051)
  AUTHORS   Voutev R, Hubbard EJ
  TITLE     A ""FLP-Out"" system for controlled gene expression in 
            Caenorhabditis elegans.
  JOURNAL   Genetics. 2008 Sep;180(1):103-19. doi: 10.1534/genetics.108.090274.
  PUBMED    18723890
REFERENCE   2  (bases 1 to 6051)
  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"; doi: 
            "10.1534/genetics.108"; journalName: "Genetics"; date: "2008-09"; 
            volume: "180"; issue: "1"; pages: "103-19"
COMMENT     Sequence Label: pGC249
FEATURES             Location/Qualifiers
     source          1..6051
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    91..214
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(255..560)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(357..376)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(902..1561)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     1476..1495
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(1615..1621,1622..1639,1640..1645))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(1671..1794)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(1819..1866)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             1942..1962
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1984..2004
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2026..2046
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2068..2088
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(2131..2159)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     2907..2927
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     primer_bind     complement(3491..3513)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3613..3632
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3710..3728)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3796..3900
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3901..3969,3970..4761)
                     /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(4119..4138)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4932..5520
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5421..5440
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5674..5691
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5808..5829
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5844..5849,5850..5867,5868..5874)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5882..5898
                     /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     5887..5909
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5906..5922
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5906..5922
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aagcttgcat gcctgcaggt cgactctaga ccgcggtttt ggcgcggatc cactagttct
       61 agcttcaaaa gcctcattac gccaagctat caactttgta tagaaaagtt gaacgagaaa
      121 cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa cagactacat
      181 aatactgtaa aacacaacat atccagtcac tatggtcgac ctgcagactg gctgtgtata
      241 agggagcctg acatttatat tccccagaac atcaggttaa tggcgttttt gatgtcattt
      301 tcgcggtggc tgagatcagc cacttcttcc ccgataacgg agaccggcac actggccata
      361 tcggtggtca tcatgcgcca gctttcatcc ccgatatgca ccaccgggta aagttcacgg
      421 gggactttat ctgacagcag acgtgcactg gccaggggga tcaccatccg tcgcccgggc
      481 gtgtcaataa tatcactctg tacatccaca aacagacgat aacggctctc tcttttatag
      541 gtgtaaacct taaactgcat ttcaccagcc cctgttctcg tcggcaaaag agccgttcat
      601 ttcaataaac cgggcgacct cagccatccc ttcctgattt tccgctttcc agcgttcggc
      661 acgcagacga cgggcttcat tctgcatggt tgtgcttacc gaaccggaga tattgacatc
      721 atatatgcct tgagcaactg atagctgtcg ctgtcaactg tcactgtaat acgctgcttc
      781 atagcatacc tctttttgac atacttcggg tatacatatc agtatatatt cttataccgc
      841 aaaaatcagc gcgcaaatac gcatactgtt atctggcttt tagtaagccg gatcctctag
      901 attacgcccc gccctgccac tcatcgcagt actgttgtaa ttcattaagc attctgccga
      961 catggaagcc atcacaaacg gcatgatgaa cctgaatcgc cagcggcatc agcaccttgt
     1021 cgccttgcgt ataatatttg cccatggtga aaacgggggc gaagaagttg tccatattgg
     1081 ccacgtttaa atcaaaactg gtgaaactca cccagggatt ggctgagacg aaaaacatat
     1141 tctcaataaa ccctttaggg aaataggcca ggttttcacc gtaacacgcc acatcttgcg
     1201 aatatatgtg tagaaactgc cggaaatcgt cgtggtattc actccagagc gatgaaaacg
     1261 tttcagtttg ctcatggaaa acggtgtaac aagggtgaac actatcccat atcaccagct
     1321 caccgtcttt cattgccata cggaattccg gatgagcatt catcaggcgg gcaagaatgt
     1381 gaataaaggc cggataaaac ttgtgcttat ttttctttac ggtctttaaa aaggccgtaa
     1441 tatccagctg aacggtctgg ttataggtac attgagcaac tgactgaaat gcctcaaaat
     1501 gttctttacg atgccattgg gatatatcaa cggtggtata tccagtgatt tttttctcca
     1561 ttttagcttc cttagctcct gaaaatctcg acggatccta actcaaaatc cacacattat
     1621 acgagccgga agcataaagt gtaaagcctg ggggtgccta atgcggccgc catagtgact
     1681 ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata
     1741 tattgatatt tatatcattt tacgtttctc gttcaacttt attatacata gttgataatt
     1801 cactgggcta gccgctctga agttcctata ctttctagag aataggaact tcggaatagg
     1861 aacttcaaga tccccgggta tgtttcgaat gatactaaca taacatagaa cattttcagg
     1921 agctcagaaa aaatgactgc tccaaagaag aagcgtaagg taccggcaga aaaaatgact
     1981 gctccaaaga agaagcgtaa ggtaccggca gaaaaaatga ctgctccaaa gaagaagcgt
     2041 aaggtaccgg cagaaaaaat gactgctcca aagaagaagc gtaaggtacc ggtagaaaaa
     2101 atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt
     2161 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga
     2221 aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg ggtaagttta
     2281 aacatatata tactaactaa ccctgattat ttaaattttc agccaacact tgtcactact
     2341 ttctgttatg gtgttcaatg cttctcgaga tacccagatc atatgaaacg gcatgacttt
     2401 ttcaagagtg ccatgcccga aggttatgta caggaaagaa ctatattttt caaagatgac
     2461 gggaactaca agacacgtaa gtttaaacag ttcggtacta actaaccata catatttaaa
     2521 ttttcaggtg ctgaagtcaa gtttgaaggt gatacccttg ttaatagaat cgagttaaaa
     2581 ggtattgatt ttaaagaaga tggaaacatt cttggacaca aattggaata caactataac
     2641 tcacacaatg tatacatcat ggcagacaaa caaaagaatg gaatcaaagt tgtaagttta
     2701 aacatgattt tactaactaa ctaatctgat ttaaattttc agaacttcaa aattagacac
     2761 aacattgaag atggaagcgt tcaactagca gaccattatc aacaaaatac tccaattggc
     2821 gatggccctg tccttttacc agacaaccat tacctgtcca cacaatctgc cctttcgaaa
     2881 gatcccaacg aaaagagaga ccacatggtc cttcttgagt ttgtaacagc tgctgggatt
     2941 acacatggca tggacgaact atacaaaagc ccacaagctt tggaattcgc tagccggcca
     3001 tacaagtaat ccggatgatc gacgccaacg tcgttgaatt ttcaaatttt aaatactgaa
     3061 tatttgtttt ttttcctatt atttatttat tctctttgtg ttttttttct tgctttctaa
     3121 aaaattaatt caatccaaat ctaaacatga gcggtttttt ttctctttcc gtctcccaat
     3181 tcgtattccg ctcctctcat ctgaacacaa tgtgcaagtt tatttatctt ctcgctttca
     3241 tttcattagg acgtgggggg aattggtgga agggggaaac acacaaaagg atgatggaaa
     3301 tgaaataagg acacacaata tgcaacaaca ttcaattcag aaatatggag gaaggtttaa
     3361 aagaaaacat aaaaatatat agaggaggaa ggaaaactag taaaaaataa gcaaagaaat
     3421 taggcgaacg atgagaattg tcctcgcttg ggccctttcg tctcgcgcgt ttcggtgatg
     3481 acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg
     3541 atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct
     3601 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
     3661 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg gccttaaggg cctcgtgata
     3721 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     3781 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3841 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3901 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3961 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     4021 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     4081 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     4141 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     4201 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     4261 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     4321 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     4381 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     4441 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     4501 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     4561 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     4621 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4681 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4741 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4801 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4861 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4921 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4981 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     5041 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     5101 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     5161 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     5221 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     5281 gagcgaacga cctacaccga actgagatac ctacagcgtg agcattgaga aagcgccacg
     5341 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     5401 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     5461 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     5521 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     5581 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     5641 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     5701 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcagctgg
     5761 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaattaa tgtgagttag
     5821 ctcactcatt aggcacccca ggctttacac tttatgcttc cggctcgtat gttgtgtgga
     5881 attgtgagcg gataacaatt tcacacagga aacagctatg accatgatta cgccaagctg
     5941 taagtttaaa catgatctta ctaactaact attctcattt aaattttcag agcttaaaaa
     6001 tggctgaaat cactcacaac gatggatacg ctaacaactt ggaaatgaaa t
//
