LOCUS       sequence-203179-        6868 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination vector for gateway cloning. Developed for Flp-In T-REx 
            system..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6868)
  AUTHORS   Yamaji M, Jishage M, Meyer C, Suryawanshi H, Der E, Yamaji M, Garzia
            A, Morozov P, Manickavel S, McFarland HL, Roeder RG, Hafner M, 
            Tuschl T
  TITLE     DND1 maintains germline stem cells via recruitment of the CCR4-NOT 
            complex to target mRNAs.
  JOURNAL   Nature. 2017 Mar 23;543(7646):568-572. doi: 10.1038/nature21690. 
            Epub 2017 Mar 15.
  PUBMED    28297718
REFERENCE   2  (bases 1 to 6868)
  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: 
            "10.1038/nature21690"; journalName: "Nature"; date: "2017-03-23- 
            23"; volume: "543"; issue: "7646"; pages: "568-572"
COMMENT     Sequence Label: pFRT-TO-DEST
FEATURES             Location/Qualifiers
     source          1..6868
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    750..874
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(899..904,905..922,923..929)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             983..1642
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1049..1068)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1984..2289
                     /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
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     2168..2187
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2330..2454)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2507..2548
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2586..2603)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2592..2816
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2949..2968)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3100..3147
                     /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             3155..4177
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3155..4177
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4307..4428
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4344..4363)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4398..4417
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4477..4493)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4477..4493)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4490..4512)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4501..4517
                     /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(4525..4531,4532..4549,4550..4555))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4570..4591
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4708..4725)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4879..5467)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4959..4978)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5638..6429,6430..6498))
                     /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     6261..6280
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6499..6603)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6678..6697)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagctta caagtttgta caaaaaagct gaacgagaaa
      781 cgtaaaatga tataaatatc aatatattaa attagatttt gcataaaaaa cagactacat
      841 aatactgtaa aacacaacat atccagtcac tatggcggcc gcattaggca ccccaggctt
      901 tacactttat gcttccggct cgtataatgt gtggattttg agttaggatc cgtcgagatt
      961 ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat
     1021 atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta
     1081 taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca
     1141 caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt
     1201 ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac
     1261 cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt
     1321 ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta
     1381 tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt
     1441 caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat
     1501 gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca
     1561 tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga
     1621 tgagtggcag ggcggggcgt aaagatctgg atccggctta ctaaaagcca gataacagta
     1681 tgcgtatttg cgcgctgatt tttgcggtat aagaatatat actgatatgt atacccgaag
     1741 tatgtcaaaa agaggtatgc tatgaagcag cgtattacag tgacagttga cagcgacagc
     1801 tatcagttgc tcaaggcata tatgatgtca atatctccgg tctggtaagc acaaccatgc
     1861 agaatgaagc ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag gaagggatgg
     1921 ctgaggtcgc ccggtttatt gaaatgaacg gctcttttgc tgacgagaac aggggctggt
     1981 gaaatgcagt ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat
     2041 gtacagagtg atattattga cacgcccggg cgacggatgg tgatccccct ggccagtgca
     2101 cgtctgctgt cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa
     2161 agctggcgca tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa
     2221 gtggctgatc tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg
     2281 ggaatataaa tgtcaggctc ccttatacac agccagtctg caggtcgacc atagtgactg
     2341 gatatgttgt gttttacagt attatgtagt ctgtttttta tgcaaaatct aatttaatat
     2401 attgatattt atatcatttt acgtttctcg ttcagctttc ttgtacaaag tggttgatat
     2461 ccagcacagt ggcggccgct caagtctaga gggcccgcgg ttcgaaggta agcctatccc
     2521 taaccctctc ctcggtctcg attctacgct cgagtctaga gggcccgttt aaacccgctg
     2581 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
     2641 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
     2701 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa
     2761 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc
     2821 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc
     2881 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct
     2941 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg
     3001 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga
     3061 ccccaaaaaa cttgattagg gtgatggttc acgtacctag aagttcctat tccgaagttc
     3121 ctattctcta gaaagtatag gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc
     3181 tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga
     3241 gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt
     3301 aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc
     3361 cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg
     3421 catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc
     3481 tgttctgcag ccggtcgcgg aggccatgga tgcgatcgct gcggccgatc ttagccagac
     3541 gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt
     3601 catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt
     3661 cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga
     3721 agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg
     3781 cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc
     3841 caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga
     3901 gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg
     3961 tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca
     4021 gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc
     4081 ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa
     4141 ccgacgcccc agcactcgtc cgagggcaaa ggaatagcac gtactacgag atttcgattc
     4201 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     4261 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc
     4321 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4381 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat
     4441 accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     4501 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     4561 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     4621 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4681 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4741 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4801 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4861 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4921 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4981 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5041 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5101 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5161 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5221 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5281 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5341 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5401 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5461 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5521 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5581 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5641 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5701 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5761 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5821 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5881 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5941 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6001 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6061 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6121 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6181 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6241 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6301 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6361 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6421 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6481 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6541 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6601 gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc
     6661 ctatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc
     6721 cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg
     6781 caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt
     6841 cgcgatgtac gggccagata tacgcgtt
//
