LOCUS       sequence-409588-        5409 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  attR4-attR3 Destination cassette in Tol1 backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5409)
  AUTHORS   Shin M, Male I, Beane TJ, Villefranc JA, Kok FO, Zhu LJ, Lawson ND
  TITLE     Vegfc acts through ERK to induce sprouting and differentiation of 
            trunk lymphatic progenitors.
  JOURNAL   Development. 2016 Oct 15;143(20):3785-3795. Epub 2016 Sep 12.
  PUBMED    27621059
REFERENCE   2  (bases 1 to 5409)
  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: 
            "Development."; date: "2016-10-15"; volume: "143(20)"; pages: 
            "3785-3795. Epub 2016 Sep 12"
COMMENT     Sequence Label: pToneDest
FEATURES             Location/Qualifiers
     source          1..5409
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    628..751
                     /gene="mutant version of attR"
                     /label=attR4
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(1099..1404)
                     /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     complement(1201..1220)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1724..2407)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2322..2341
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2408..2510)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2656..2779)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     polyA_signal    2801..2935
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2825..2844)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2879..2898
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3136..3152)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3188..3204)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3201..3223)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3212..3228
                     /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(3236..3242,3243..3260,3261..3266))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3281..3302
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3419..3436)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3590..4178)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3670..3689)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4349..5140,5141..5209))
                     /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     4972..4991
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5210..5314)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5382..5400
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ccctttagcc agtagcggtt
      421 ctaggcacgg gccgtccggg cggtggcctg gggcggaaaa ctgaaggggg gcggcaccgg
      481 cggctcagcc ctttgtaata tattaatatg caccactatt ggtttactta tgtcacagtt
      541 tgtaagtttg taacagcctg aacctggccg cgcgccattg ggatggaacc ctcgaggggc
      601 ccgctagcga tatccctgca gggctatcaa ctttgtatag aaaagttgaa cgagaaacgt
      661 aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag actacataat
      721 actgtaaaac acaacatatc cagtcactat ggtcgaccga cagccttcca aatgttcttc
      781 tcaaacggaa tcgtcgtatc cagcctactc gctattgtcc tcaatgccgt attaaatcat
      841 aaaaagaaat aagaaaaaga ggtgcgagcc tcttttttgt gtgacaaaat aaaaacatct
      901 acctattcat atacgctagt gtcatagtcc tgaaaatcat ctgcatcaag aacaatttca
      961 caactcttat acttttctct tacaagtcgt tcggcttcat ctggattttc agcctctata
     1021 cttactaaac gtgataaagt ttctgtaatt tctactgtat cgacctgcag actggctgtg
     1081 tataagggag cctgacattt atattcccca gaacatcagg ttaatggcgt ttttgatgtc
     1141 attttcgcgg tggctgagat cagccacttc ttccccgata acggagaccg gcacactggc
     1201 catatcggtg gtcatcatgc gccagctttc atccccgata tgcaccaccg ggtaaagttc
     1261 acgggagact ttatctgaca gcagacgtgc actggccagg gggatcacca tccgtcgccc
     1321 gggcgtgtca ataatatcac tctgtacatc cacaaacaga cgataacggc tctctctttt
     1381 ataggtgtaa accttaaact gcatttcacc agcccctgtt ctcgtcagca aaagagccgt
     1441 tcatttcaat aaaccgggcg acctcagcca tcccttcctg attttccgct ttccagcgtt
     1501 cggcacgcag acgacgggct tcattctgca tggttgtgct taccagaccg gagatattga
     1561 catcatatat gccttgagca actgatagct gtcgctgtca actgtcactg taatacgctg
     1621 cttcatagca tacctctttt tgacatactt cgggtataca tatcagtata tattcttata
     1681 ccgcaaaaat cagcgcgcaa atacgcatac tgttatctgg cttttagtaa gccggatcca
     1741 cgcggcgttt acgccccccc tgccactcat cgcagtactg ttgtaattca ttaagcattc
     1801 tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc ggcatcagca
     1861 ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag aagttgtcca
     1921 tattggccac gtttaaatca aaactggtga aactcaccca gggattggct gagacgaaaa
     1981 acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa cacgccacat
     2041 cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc cagagcgatg
     2101 aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta tcccatatca
     2161 ccagctcacc gtctttcatt gccatacgga attccggatg agcattcatc aggcgggcaa
     2221 gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc tttaaaaagg
     2281 ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac tgaaatgcct
     2341 caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca gtgatttttt
     2401 tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat acgcccggta
     2461 gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca acgtctcatt
     2521 ttcgccaaaa gttggcccag ggcttcccgg tatcaacagg gacaccagga tttatttatt
     2581 ctgcgaagtg atcttccgtc acaggtattt attcggcgca aagtgcgtcg ggtgatgctg
     2641 ccaacttacg gccgccatag tgactggata tgttgtgttt tacagtatta tgtagtctgt
     2701 tttttatgca aaatctaatt taatatattg atatttatat cattttacgt ttctcgttca
     2761 actttattat acatagttga taaggcgcgc caggcctgta gatccagaca tgataagata
     2821 cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga
     2881 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     2941 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaatt
     3001 aaaacatttc agttgacgaa gacaaacaaa gttctgttgt gactatgggg ggggggggcg
     3061 cctggggatg gtctcgcccg gggagtaatt cagggtagaa ccgccactgg ctaaaggaag
     3121 cttggcgtaa tcatggtcat agctgtttcc tgtatcccaa tggcgcgccg agcttggcgt
     3181 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     3241 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     3301 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     3361 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     3421 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     3481 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     3541 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     3601 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     3661 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     3721 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3781 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3841 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3901 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3961 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4021 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4081 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4141 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4201 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     4261 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     4321 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     4381 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     4441 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     4501 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     4561 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     4621 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     4681 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     4741 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4801 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4861 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4921 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4981 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5041 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5101 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5161 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     5221 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     5281 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     5341 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     5401 cctttcgtc
//
