LOCUS       Exported                6125 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pFRT/FLAG/HA-DEST LIN28B
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6125)
  TITLE     Identification of mRNAs bound and regulated by human LIN28 proteins 
            and molecular requirements for RNA recognition
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6125)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6125
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             1..42
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(86..105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        277..656
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        657..860
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     811..831
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    862..880
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    883..901
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     911..935
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1035..1058
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1059..1085
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     protein_bind    1101..1125
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1879..1903)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1956..1997
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2077..2094)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2083..2307
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2440..2459)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2591..2638
                     /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             2646..3668
                     /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    3798..3919
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3835..3854)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3889..3908
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3968..3984)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3968..3984)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3981..4003)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3992..4008
                     /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(4016..4046)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4061..4082
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4199..4216)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4370..4958)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4450..4469)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5129..5989)
                     /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     5752..5771
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5990..6094)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtaagccta tccctaaccc tctcctcggt ctcgattcta cggacggatc gggagatctc
       61 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
      121 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
      181 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
      241 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
      301 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      361 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      421 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      481 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      541 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      601 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      661 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      721 gtctccaccc cattgacgtc aatgggagtt tgttttggaa ccaaaatcaa cgggactttc
      781 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      841 aggtctatat aagcagagct ctccctatca gtgatagaga tctccctatc agtgatagag
      901 atcgtcgacg agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt
      961 ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagcg tttaaactta
     1021 agcttggtac catggactac aaggacgacg atgacaagta cccttatgac gtgcccgatt
     1081 acgctttgaa ccagatatca acaagtttgt acaaaaaagc aggctatggc cgaaggcggg
     1141 gctagcaaag gtggtggaga agagcccggg aagctgccgg agccggcaga ggaggaatcc
     1201 caggttttgc gcggaactgg ccactgtaag tggttcaatg tgcgcatggg atttggattc
     1261 atctccatga taaaccgaga gggaagcccc ttggatattc cagtcgatgt atttgtacac
     1321 caaagcaaac tattcatgga aggatttaga agcctaaaag aaggagaacc agtggaattc
     1381 acatttaaaa aatcttccaa aggccttgag tcaatacggg taacaggacc tggtgggagc
     1441 ccctgtttag gaagtgaaag aagacccaaa gggaagacac tacagaaaag aaaaccaaag
     1501 ggagatagat gctacaactg tggtggcctt gatcatcatg ctaaggaatg tagtctacct
     1561 cctcagccaa agaagtgcca ttactgtcag agcatcatgc acatggtggc aaactgccca
     1621 cataaaaatg ttgcacagcc acccgcgagt tctcagggaa gacaggaagc agaatcccag
     1681 ccatgcactt caactctccc tcgagaagtg ggaggcgggc atggctgtac atcaccaccg
     1741 tttcctcagg aggctagggc agagatctca gaacggtcag gcaggtcacc tcaagaagct
     1801 tcctccacga agtcatctat agcaccagaa gagcaaagca aaaaggggcc ttcagttcaa
     1861 aaaaggaaaa agacataaac ccagctttct tgtacaaagt ggttgatatc cagcacagtg
     1921 gcggccgctc aagtctagag ggcccgcggt tcgaaggtaa gcctatccct aaccctctcc
     1981 tcggtctcga ttctacgcgt accggatccg cggccgcata gataactgat ccagtgtgcc
     2041 tcgagtctag agggcccgtt taaacccgct gatcagcctc gactgtgcct tctagttgcc
     2101 agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca
     2161 ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta
     2221 ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc
     2281 atgctgggga tgcggtgggc tctatggctt ctgaggcgga aagaaccagc tggggctcta
     2341 gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     2401 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     2461 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     2521 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     2581 cacgtaccta gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttcct
     2641 tggccaaaaa gcctgaactc accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg
     2701 acagcgtctc cgacctgatg cagctctcgg agggcgaaga atctcgtgct ttcagcttcg
     2761 atgtaggagg gcgtggatat gtcctgcggg taaatagctg cgccgatggt ttctacaaag
     2821 atcgttatgt ttatcggcac tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca
     2881 ttggggaatt cagcgagagc ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt
     2941 tgcaagacct gcctgaaacc gaactgcccg ctgttctgca gccggtcgcg gaggccatgg
     3001 atgcgatcgc tgcggccgat cttagccaga cgagcgggtt cggcccattc ggaccgcaag
     3061 gaatcggtca atacactaca tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt
     3121 atcactggca aactgtgatg gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg
     3181 agctgatgct ttgggccgag gactgccccg aagtccggca cctcgtgcac gcggatttcg
     3241 gctccaacaa tgtcctgacg gacaatggcc gcataacagc ggtcattgac tggagcgagg
     3301 cgatgttcgg ggattcccaa tacgaggtcg ccaacatctt cttctggagg ccgtggttgg
     3361 cttgtatgga gcagcagacg cgctacttcg agcggaggca tccggagctt gcaggatcgc
     3421 cgcggctccg ggcgtatatg ctccgcattg gtcttgacca actctatcag agcttggttg
     3481 acggcaattt cgatgatgca gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg
     3541 gagccgggac tgtcgggcgt acacaaatcg cccgcagaag cgcggccgtc tggaccgatg
     3601 gctgtgtaga agtactcgcc gatagtggaa accgacgccc cagcactcgt ccgagggcaa
     3661 aggaatagca cgtactacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     3721 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     3781 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     3841 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     3901 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     3961 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     4021 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     4081 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     4141 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     4201 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     4261 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     4321 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     4381 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     4441 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     4501 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     4561 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     4621 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     4681 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     4741 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     4801 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     4861 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     4921 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     4981 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     5041 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     5101 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     5161 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     5221 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     5281 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     5341 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     5401 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     5461 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     5521 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     5581 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     5641 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     5701 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     5761 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     5821 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     5881 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     5941 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     6001 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     6061 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     6121 acgtc
//
