LOCUS       Exported                5404 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    p3xFLAG-EYFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5404)
  AUTHORS   Lee K, Gjorevski N, Boghaert E, Radisky DC, Nelson CM
  TITLE     Snail1, Snail2, and E47 promote mammary epithelial branching 
            morphogenesis.
  JOURNAL   EMBO J. 2011 May 24;30(13):2662-74. doi: 10.1038/emboj.2011.159.
  PUBMED    21610693
REFERENCE   2  (bases 1 to 5404)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5404
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     126..148
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     140..157
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     141..157
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     enhancer        318..697
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        698..901
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     852..872
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             931..996
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1015..1737
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGM?ELYKS"
     primer_bind     complement(1060..1081)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1321..1340)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1668..1689
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1745..2367
                     /label=hGH poly(A) signal
                     /note="human growth hormone polyadenylation signal"
     primer_bind     complement(1944..1963)
                     /label=hGH-PA-R
                     /note="Human growth hormone terminator, reverse primer"
     primer_bind     complement(2391..2411)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2396..2725
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2576..2711
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2638..2657
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(2763..2782)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2764..2782)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2796..2812)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2796..2812)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2809..2831)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2820..2836
                     /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(2844..2874)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2889..2910
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3027..3044)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3198..3786)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3278..3297)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(3957..4817)
                     /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     4580..4599
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4818..4922)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4949..5404
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5036..5055)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5246..5267
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct
       61 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg
      121 gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc aagctgatct atacattgaa
      181 tcaatattgg caattagcca tattagtcat tggttatata gcataaatca atattggcta
      241 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc
      301 aatatgaccg ccatgttgac attgattatt gactagttat taatagtaat caattacggg
      361 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      421 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      481 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      541 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga
      601 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg
      661 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac
      721 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      781 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc
      841 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc
      901 tcgtttagtg aaccgtcaga attaaccatg gactacaaag accatgacgg tgattataaa
      961 gatcatgaca tcgattacaa ggatgacgat gacaagcttg cggccgcgaa ttcaatggtg
     1021 agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac
     1081 gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag
     1141 ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg
     1201 accaccttcg gctacggcct gcagtgcttc gcccgctacc ccgaccacat gaagcagcac
     1261 gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag
     1321 gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac
     1381 cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg
     1441 gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc
     1501 aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac
     1561 taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg
     1621 agctaccagt ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg
     1681 gagttcgtga ccgccgccgg gatcactctc ggcatggncg agctgtacaa gtcctgagga
     1741 tcccgggtgg catccctgtg acccctcccc agtgcctctc ctggccctgg aagttgccac
     1801 tccagtgccc accagccttg tcctaataaa attaagttgc atcattttgt ctgactaggt
     1861 gtccttctat aatattatgg ggtggagggg ggtggtatgg agcaaggggc aagttgggaa
     1921 gacaacctgt agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc
     1981 ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga
     2041 gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt tttggtagag
     2101 acggggtttc accatattgg ccaggctggt ctccaactcc taatctcagg tgatctaccc
     2161 accttggcct cccaaattgc tgggattaca ggcgtgaacc actgctccct tccctgtcct
     2221 tctgatttta aaataactat accagcagga ggacgtccag acacagcata ggctacctgg
     2281 ccatgcccaa ccggtgggac atttgagttg cttgcttggc actgtcctct catgcgttgg
     2341 gtccactcag tagatgcctg ttgaattggg tacgcggcca gcttggctgt ggaatgtgtg
     2401 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2461 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2521 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     2581 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2641 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     2701 ttttggaggc ctaggctttt gcaaaaagct cctcgaggaa ctgaaaaacc agaaagttaa
     2761 ttccctatag tgagtcgtat taaattcgta atcatgtcat agctgtttcc tgtgtgaaat
     2821 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     2881 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     2941 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3001 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3061 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3121 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3181 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3241 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3301 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3361 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3421 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3481 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3541 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3601 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3661 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3721 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3781 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3841 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3901 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3961 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4021 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4081 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4141 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4201 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4261 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4321 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4381 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4441 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4501 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4561 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4621 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4681 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4741 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4801 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4861 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4921 cgcacatttc cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg
     4981 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     5041 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     5101 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     5161 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     5221 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     5281 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     5341 aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgcttaca
     5401 attt
//
