LOCUS       V015817                 6846 bp    DNA     circular SYN 23-OCT-2019
DEFINITION  Exported.
ACCESSION   V015817
VERSION     V015817
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 6846)
  AUTHORS   Stach C, McCann M, O'Brien C, Le TS, Somia N, Chen X, Lee K, Fu HY,
            Daoutidis P, Zhao L, Hu WS, Smanski MJ.
  TITLE     Model-driven engineering of N-linked glycosylation in Chinese
            Hamster Ovary cells
  JOURNAL   ACS Synth Biol (2019) In press
   PUBMED   31596566
REFERENCE   2  (bases 1 to 6846)
  AUTHORS   Stach CS.
  TITLE     Direct Submission
  JOURNAL   Submitted (30-APR-2019) Biochemistry, Molecular Biology, and
            Biophysics; Chemical Engineering and Materials Science, University
            of Minnesota, 1479 Gortner Ave, Suite 344, Saint Paul, Minnesota
            55108, United States
REFERENCE   3  (bases 1 to 6846)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing
            ##Assembly-Data-END##
            SGRef: number: 1; type: "Journal Article"; journalName: "ACS Synth
            Biol (2019) In press"
            SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
            (30-APR-2019) Biochemistry, Molecular Biology, and Biophysics;
            Chemical Engineering and Materials Science, University of Minnesota,
            1479 Gortner Ave, Suite 344, Saint Paul, Minnesota 55108, United
            States"
FEATURES             Location/Qualifiers
     source          1..6846
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    21..54
                     /label="lox2272"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are
                     compatible with each other, but incompatible with loxP or
                     loxN sites (Lee and Saito, 1988)."
     protein_bind    complement(55..88)
                     /label="FRT (minimal)"
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
     misc_feature    89..94
                     /label="BbsI site"
                     /note="BbsI site"
     misc_feature    97..100
                     /label="Golden gate cloning scar"
                     /note="Golden gate cloning scar"
     misc_feature    105..111
                     /label="AarI site"
                     /note="AarI site"
     primer_bind     313..329
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     misc_feature    330..386
                     /label="MCS"
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(399..415)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(423..439)
                     /label="lac operator"
                     /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(447..477)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(492..513)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    594..600
                     /label="AarI site"
                     /note="AarI site"
     misc_feature    605..608
                     /label="Golden gate cloning scar"
                     /note="Golden gate cloning scar"
     misc_feature    611..616
                     /label="BbsI site"
                     /note="BbsI site"
     promoter        622..1833
                     /label="UbC promoter"
                     /note="human ubiquitin C promoter"
     CDS             1865..2284
                     /gene="bsr"
                     /label="Blasticidin-S deaminase"
                     /note="Blasticidin-S deaminase from Bacillus cereus.
                     Accession#: P33967"
     polyA_signal    2334..2382
                     /label="poly(A) signal"
                     /note="synthetic polyadenylation signal"
     protein_bind    2383..2416
                     /label="lox5171"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (ATGTGTAC) (Shaw et al., 2021). lox5171 sites are
                     compatible with each other, but incompatible with loxP or
                     loxN sites (Lee and Saito, 1988)."
     misc_feature    2417..2450
                     /label="F3"
                     /note="F3"
     misc_feature    2469..2507
                     /label="PhiC31 attP"
                     /note="PhiC31 attP"
     misc_feature    2697
                     /label="mutation to remove SapI site"
                     /note="mutation to remove SapI site"
     CDS             complement(2833..3690)
                     /label="AmpR"
                     /note="beta-lactamase"
     terminator      4223..4309
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      4401..4428
                     /label="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     rep_origin      4562..5150
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     misc_feature    5330
                     /label="mutation to remove SapI site"
                     /note="mutation to remove SapI site"
     misc_feature    complement(5336..5476)
                     /label="bom"
                     /note="basis of mobility region from pBR322"
     CDS             complement(6773..6790)
                     /label="6xHis"
                     /note="6xHis affinity tag"
     protein_bind    6811..6844
                     /label="attB"
                     /note="minimal attB site for the phi-C31 integrase (Groth
                     et al., 2000)"
ORIGIN
        1 gctacgctga ctgcagtgca ataacttcgt ataaagtatc ctatacgaag ttatgaagtt
       61 cctatacttt ctagagaata ggaacttcga agacagggag gtaagcaggt gtgcaccata
      121 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc
      181 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
      241 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
      301 agtcacgacg ttgtaaaacg acggccagtg aattcgagct cggtacccgg ggatcctcta
      361 gagtcgacct gcaggcatgc aagcttggcg taatcatggt catagctgtt tcctgtgtga
      421 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
      481 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
      541 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc gggcacctgc
      601 ttactactct gtcttcgatc tggcctccgc gccgggtttt ggcgcctccc gcgggcgccc
      661 ccctcctcac ggcgagcgct gccacgtcag acgaagggcg cagcgagcgt cctgatcctt
      721 ccgcccggac gctcaggaca gcggcccgct gctcataaga ctcggcctta gaaccccagt
      781 atcagcagaa ggacatttta ggacgggact tgggtgactc tagggcactg gttttctttc
      841 cagagagcgg aacaggcgag gaaaagtagt cccttctcgg cgattctgcg gagggatctc
      901 cgtggggcgg tgaacgccga tgattatata aggacgcgcc gggtgtggca cagctagttc
      961 cgtcgcagcc gggatttggg tcgcggttct tgtttgtgga tcgctgtgat cgtcacttgg
     1021 tgagtagcgg gctgctgggc tggccggggc tttcgtggcc gccgggccgc tcggtgggac
     1081 ggaagcgtgt ggagagaccg ccaagggctg tagtctgggt ccgcgagcaa ggttgccctg
     1141 aactgggggt tggggggagc gcagcaaaat ggcggctgtt cccgagtctt gaatggagga
     1201 cgcttgtgag gcgggctgtg aggtcgttga aacaaggtgg ggggcatggt gggcggcaag
     1261 aacccaaggt cttgaggcct tcgctaatgc gggaaagctc ttattcgggt gagatgggct
     1321 ggggcaccat ctggggaccc tgacgtgaag tttgtcactg actggagaac tcggtttgtc
     1381 gtctgttgcg ggggcggcag ttatggcggt gccgttgggc agtgcacccg tacctttggg
     1441 agcgcgcgcc ctcgtcgtgt cgtgacgtca cccgttctgt tggcttataa tgcagggtgg
     1501 ggccacgtgc cggtaggtgt gcggtaggct tttctccgtc gcaggacgca gggttcgggc
     1561 ctagggtagg ctctcctgaa tcgacaggcg ccggacctct ggtgagggga gggataagtg
     1621 aggcgtcagt ttctttggtc ggttttatgt acctatcttc ttaagtagct gaagctccgg
     1681 ttttgaacta tgcgctcggg gttggcgagt gtgttttgtg aagtttttta ggcacctttt
     1741 gaaatgtaat catttgggtc aatatgtaat tttcagtgtt agactagtaa attgtccgct
     1801 aaattctggc cgtttttggc ttttttgtta gacgaagctt gggcccggga attaattcgc
     1861 caccatggaa accttcaaca tctctcagca ggatctggag ctggtggagg tcgccactga
     1921 gaagatcacc atgctctatg aggacaacaa gcaccatgtc ggggcggcca tcaggaccaa
     1981 gactggggag atcatctctg ctgtccacat tgaagcctac attggcaggg tcactgtctg
     2041 tgctgaagcc attgccattg ggtctgctgt gagcaacggg cagaaggact ttgacaccat
     2101 tgtggctgtc aggcacccct actctgatga ggtggacaga tccatcaggg tggtcagccc
     2161 ctgtggcatg tgtagagagc tgatctctga ctatgctcct gactgctttg tgctcattga
     2221 gatgaatggc aagctggtca aaaccaccat tgaggaactc atccccctca agtacaccag
     2281 gaactaaacc tgagctagct tgactgactg agatacagcg taccttcagc tcaaataaaa
     2341 tatctttatt ttcattacat ctgtgtgttg gttttttgtg tgataacttc gtataatgtg
     2401 tactatacga agttatgaag ttcctatact atttgaagaa taggaacttc tcgacccatg
     2461 ggggcccgcc ccaactgggg taacctttga gttctctcag ttgggggtaa tcagcatcat
     2521 gatgtggtac cacatcatga tgctgattat aagaatgcgg ccgccacact ctagtggatc
     2581 tcgagttaat aattcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat
     2641 cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagccctt
     2701 cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc
     2761 cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat
     2821 cgccatgggt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     2881 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     2941 atctggcccc agtgctgcaa tgataccgcg agagccacgc tcaccggctc cagatttatc
     3001 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     3061 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     3121 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     3181 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     3241 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     3301 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     3361 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     3421 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     3481 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     3541 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     3601 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     3661 aagggcgaca cggaaatgtt gaatactcat gcgaaacgat cctcatcctg tctcttgatc
     3721 agagcttgat cccctgcgcc atcagatcct tggcggcgag aaagccatcc agtttacttt
     3781 gcagggcttc ccaaccttac cagagggcgc cccagctggc aattccggtt cgcttgctgt
     3841 ccataaaacc gcccagtcta gctatcgcca tgtaagccca ctgcaagcta cctgctttct
     3901 ctttgcgctt gcgttttccc ttgtccagat agcccagtag ctgacattca tccggggtca
     3961 gcaccgtttc tgcggactgg ctttctacgt gctcgagggg ggccaaacgg tctccagctt
     4021 ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc agaacgcaga
     4081 agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc
     4141 atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg
     4201 agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     4261 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc
     4321 ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac
     4381 tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca
     4441 aactcttttg tttatttttc taaatacatt caaatatgta tccgctcatg accaaaatcc
     4501 cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt
     4561 cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac
     4621 cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct
     4681 tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact
     4741 tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg
     4801 ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata
     4861 aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga
     4921 cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag
     4981 ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg
     5041 agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac
     5101 ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca
     5161 acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg
     5221 cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc
     5281 gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggag gagcgcctga
     5341 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca
     5401 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     5461 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     5521 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     5581 gaggttttca ccgtcatcac cgaaacgcgc gaggcagcag atcaattcgc gcgcgaaggc
     5641 gaagcggcat gcataatgtg cctgtcaaat ggacgaagca gggattctgc aaaccctatg
     5701 ctactccgtc aagccgtcaa ttgtctgatt cgttaccaat tatgacaact tgacggctac
     5761 atcattcact ttttcttcac aaccggcacg gaactcgctc gggctggccc cggtgcattt
     5821 tttaaatacc cgcgagaaat agagttgatc gtcaaaacca acattgcgac cgacggtggc
     5881 gataggcatc cgggtggtgc tcaaaagcag cttcgcctgg ctgatacgtt ggtcctcgcg
     5941 ccagcttaag acgctaatcc ctaactgctg gcggaaaaga tgtgacagac gcgacggcga
     6001 caagcaaaca tgctgtgcga cgctggcgat acattaccct gttatcccta gatacattac
     6061 cctgttatcc cagatgacat accctgttat ccctagatga cattaccctg ttatcccaga
     6121 tgacattacc ctgttatccc tagatacatt accctgttat cccagatgac ataccctgtt
     6181 atccctagat gacattaccc tgttatccca gatgacatta ccctgttatc cctagataca
     6241 ttaccctgtt atcccagatg acataccctg ttatccctag atgacattac cctgttatcc
     6301 cagatgacat taccctgtta tccctagata cattaccctg ttatcccaga tgacataccc
     6361 tgttatccct agatgacatt accctgttat cccagatgac attaccctgt tatccctaga
     6421 tacattaccc tgttatccca gatgacatac cctgttatcc ctagatgaca ttaccctgtt
     6481 atcccagatg acattaccct gttatcccta gatacattac cctgttatcc cagatgacat
     6541 accctgttat ccctagatga cattaccctg ttatcccaga tgacattacc ctgttatccc
     6601 tagatacatt accctgttat cccagatgac ataccctgtt atccctagat gacattaccc
     6661 tgttatccca gatgacatta ccctgttatc cctagataca ttaccctgtt atcccagatg
     6721 acataccctg ttatccctag atgacattac cctgttatcc cagataaact caatgatgat
     6781 gatgatgatg gtcgagactc agcggccgcg gtgccagggc gtgcccttgg gctccccggg
     6841 cgcgac
//
