LOCUS       V015655                 6435 bp    DNA     circular SYN 23-OCT-2019
DEFINITION  Exported.
ACCESSION   V015655
VERSION     V015655
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 6435)
  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 6435)
  AUTHORS   Stach CS.
  TITLE     Direct Submission
  JOURNAL   Submitted (28-AUG-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 6435)
  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
            (28-AUG-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..6435
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(21..54)
                     /label="FRT (minimal)"
                     /note="supports FLP-mediated excision but not integration
                     (Turan and Bode, 2011)"
     misc_feature    55..65
                     /label="AarI site"
                     /note="AarI site"
     misc_feature    66..69
                     /label="Golden gate cloning scar"
                     /note="Golden gate cloning scar"
     misc_feature    70..77
                     /label="BbsI site"
                     /note="BbsI site"
     primer_bind     279..295
                     /label="M13 fwd"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     misc_feature    296..352
                     /label="MCS"
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(365..381)
                     /label="M13 rev"
                     /note="common sequencing primer, one of multiple similar
                     variants"
     protein_bind    complement(389..405)
                     /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(413..443)
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(458..479)
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     misc_feature    560..567
                     /label="BbsI site"
                     /note="BbsI site"
     misc_feature    568..571
                     /label="Golden gate cloning scar"
                     /note="Golden gate cloning scar"
     misc_feature    572..582
                     /label="AarI site"
                     /note="AarI site"
     promoter        588..1799
                     /label="UbC promoter"
                     /note="human ubiquitin C promoter"
     CDS             1831..2250
                     /gene="bsr"
                     /label="Blasticidin-S deaminase"
                     /note="Blasticidin-S deaminase from Bacillus cereus.
                     Accession#: P33967"
     polyA_signal    2257..2305
                     /label="poly(A) signal"
                     /note="synthetic polyadenylation signal"
     protein_bind    complement(2306..2339)
                     /label="loxP"
                     /note="Cre-mediated recombination occurs in the 8-bp core
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    2358..2396
                     /label="PhiC31 attP"
                     /note="PhiC31 attP"
     CDS             complement(2719..3279)
                     /codon_start=1
                     /transl_table=11
                     /product="kanamycin resistance protein"
                     /label="kanamycin resistance protein"
                     /protein_id="QFQ66234.1"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLHV
                     IYTLLLPRKYPSWLMQCGGCIRLIRLPAHSTTKRNIASSEHVLGWKPVLSIRMIWTKSI
                     RGSRQPNCSPGSRRACPTARISS"
     terminator      3812..3898
                     /label="rrnB T1 terminator"
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      3990..4017
                     /label="rrnB T2 terminator"
                     /note="transcription terminator T2 from the E. coli rrnB
                     gene"
     rep_origin      4151..4739
                     /label="ori"
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
     misc_feature    complement(4925..5065)
                     /label="bom"
                     /note="basis of mobility region from pBR322"
     CDS             complement(6362..6379)
                     /label="6xHis"
                     /note="6xHis affinity tag"
     protein_bind    6400..6433
                     /label="attB"
                     /note="minimal attB site for the phi-C31 integrase (Groth
                     et al., 2000)"
ORIGIN
        1 gctacgctga ctgcagtgca gaagttccta tactttctag agaataggaa cttccacctg
       61 cttacggagc tgtcttctgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
      121 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
      181 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
      241 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
      301 cgagctcggt acccggggat cctctagagt cgacctgcag gcatgcaagc ttggcgtaat
      361 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
      421 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
      481 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
      541 gaatcggcca acgcgcgggg aagacagagg tgtaagcagg tggatctggc ctccgcgccg
      601 ggttttggcg cctcccgcgg gcgcccccct cctcacggcg agcgctgcca cgtcagacga
      661 agggcgcagc gagcgtcctg atccttccgc ccggacgctc aggacagcgg cccgctgctc
      721 ataagactcg gccttagaac cccagtatca gcagaaggac attttaggac gggacttggg
      781 tgactctagg gcactggttt tctttccaga gagcggaaca ggcgaggaaa agtagtccct
      841 tctcggcgat tctgcggagg gatctccgtg gggcggtgaa cgccgatgat tatataagga
      901 cgcgccgggt gtggcacagc tagttccgtc gcagccggga tttgggtcgc ggttcttgtt
      961 tgtggatcgc tgtgatcgtc acttggtgag tagcgggctg ctgggctggc cggggctttc
     1021 gtggccgccg ggccgctcgg tgggacggaa gcgtgtggag agaccgccaa gggctgtagt
     1081 ctgggtccgc gagcaaggtt gccctgaact gggggttggg gggagcgcag caaaatggcg
     1141 gctgttcccg agtcttgaat ggaggacgct tgtgaggcgg gctgtgaggt cgttgaaaca
     1201 aggtgggggg catggtgggc ggcaagaacc caaggtcttg aggccttcgc taatgcggga
     1261 aagctcttat tcgggtgaga tgggctgggg caccatctgg ggaccctgac gtgaagtttg
     1321 tcactgactg gagaactcgg tttgtcgtct gttgcggggg cggcagttat ggcggtgccg
     1381 ttgggcagtg cacccgtacc tttgggagcg cgcgccctcg tcgtgtcgtg acgtcacccg
     1441 ttctgttggc ttataatgca gggtggggcc acctgccggt aggtgtgcgg taggcttttc
     1501 tccgtcgcag gacgcagggt tcgggcctag ggtaggctct cctgaatcga caggcgccgg
     1561 acctctggtg aggggaggga taagtgaggc gtcagtttct ttggtcggtt ttatgtacct
     1621 atcttcttaa gtagctgaag ctccggtttt gaactatgcg ctcggggttg gcgagtgtgt
     1681 tttgtgaagt tttttaggca ccttttgaaa tgtaatcatt tgggtcaata tgtaattttc
     1741 agtgttagac tagtaaattg tccgctaaat tctggccgtt tttggctttt ttgttagacg
     1801 aagcttgggc ccgggaatta attcgccacc atggaaacct tcaacatctc tcagcaggat
     1861 ctggagctgg tggaggtcgc cactgagaag atcaccatgc tctatgagga caacaagcac
     1921 catgtcgggg cggccatcag gaccaagact ggggagatca tctctgctgt ccacattgaa
     1981 gcctacattg gcagggtcac tgtctgtgct gaagccattg ccattgggtc tgctgtgagc
     2041 aacgggcaga aggactttga caccattgtg gctgtcaggc acccctactc tgatgaggtg
     2101 gacagatcca tcagggtggt cagcccctgt ggcatgtgta gagagctgat ctctgactat
     2161 gctcctgact gctttgtgct cattgagatg aatggcaagc tggtcaaaac caccattgag
     2221 gaactcatcc ccctcaagta caccaggaac taatgaaata aaatatcttt attttcatta
     2281 catctgtgtg ttggtttttt gtgtgataac ttcgtatagc atacattata cgaagttatt
     2341 cgacccatgg gggcccgccc caactggggt aacctttgag ttctctcagt tgggggtaat
     2401 cagcatcatg atgtggtacc acatcatgat gctgattata agaatgcggc cgccacactc
     2461 tagtggatct cgagttaata attcagaaga actcgtcaag aaggcgatag aaggcgatgc
     2521 gctgcgaatc gggagcggcg ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc
     2581 caagctcttc agcaatatca cgggtagcca acgctatgtc ctgatagcgg tccgccacac
     2641 ccagccggcc acagtcgatg aatccagaaa agcggccatt ttccaccatg atattcggca
     2701 agcaggcatc gccatgggtc acgacgagat cctcgccgtc gggcatgctc gccttgagcc
     2761 tggcgaacag ttcggctggc gcgagcccct gatgctcttc gtccagatca tcctgatcga
     2821 caagaccggc ttccatccga gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga
     2881 atgggcaggt agccggatca agcgtatgca gccgccgcat tgcatcagcc atgatggata
     2941 ctttctcggc aggagcaagg tgtagatgac atggagatcc tgccccggca cttcgcccaa
     3001 tagcagccag tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc
     3061 cgtcgtggcc agccacgata gccgcgctgc ctcgtcttgc agttcattca gggcaccgga
     3121 caggtcggtc ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc
     3181 atcagagcag ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc
     3241 ggccggagaa cctgcgtgca atccatcttg ttcaatcatg cgaaacgatc ctcatcctgt
     3301 ctcttgatca gagcttgatc ccctgcgcca tcagatcctt ggcggcgaga aagccatcca
     3361 gtttactttg cagggcttcc caaccttacc agagggcgcc ccagctggca attccggttc
     3421 gcttgctgtc cataaaaccg cccagtctag ctatcgccat gtaagcccac tgcaagctac
     3481 ctgctttctc tttgcgcttg cgttttccct tgtccagata gcccagtagc tgacattcat
     3541 ccggggtcag caccgtttct gcggactggc tttctacgtg ctcgaggggg gccaaacggt
     3601 ctccagcttg gctgttttgg cggatgagag aagattttca gcctgataca gattaaatca
     3661 gaacgcagaa gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca
     3721 cctgacccca tgccgaactc agaagtgaaa cgccgtagcg ccgatggtag tgtggggtct
     3781 ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga
     3841 ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc
     3901 gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc
     3961 gccataaact gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc
     4021 gtttctacaa actcttttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4081 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     4141 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4201 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4261 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     4321 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4381 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4441 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4501 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4561 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4621 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4681 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4741 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4801 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4861 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4921 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     4981 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     5041 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     5101 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     5161 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagcaga tcaattcgcg
     5221 cgcgaaggcg aagcggcatg cataatgtgc ctgtcaaatg gacgaagcag ggattctgca
     5281 aaccctatgc tactccgtca agccgtcaat tgtctgattc gttaccaatt atgacaactt
     5341 gacggctaca tcattcactt tttcttcaca accggcacgg aactcgctcg ggctggcccc
     5401 ggtgcatttt ttaaataccc gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc
     5461 gacggtggcg ataggcatcc gggtggtgct caaaagcagc ttcgcctggc tgatacgttg
     5521 gtcctcgcgc cagcttaaga cgctaatccc taactgctgg cggaaaagat gtgacagacg
     5581 cgacggcgac aagcaaacat gctgtgcgac gctggcgata cattaccctg ttatccctag
     5641 atacattacc ctgttatccc agatgacata ccctgttatc cctagatgac attaccctgt
     5701 tatcccagat gacattaccc tgttatccct agatacatta ccctgttatc ccagatgaca
     5761 taccctgtta tccctagatg acattaccct gttatcccag atgacattac cctgttatcc
     5821 ctagatacat taccctgtta tcccagatga cataccctgt tatccctaga tgacattacc
     5881 ctgttatccc agatgacatt accctgttat ccctagatac attaccctgt tatcccagat
     5941 gacataccct gttatcccta gatgacatta ccctgttatc ccagatgaca ttaccctgtt
     6001 atccctagat acattaccct gttatcccag atgacatacc ctgttatccc tagatgacat
     6061 taccctgtta tcccagatga cattaccctg ttatccctag atacattacc ctgttatccc
     6121 agatgacata ccctgttatc cctagatgac attaccctgt tatcccagat gacattaccc
     6181 tgttatccct agatacatta ccctgttatc ccagatgaca taccctgtta tccctagatg
     6241 acattaccct gttatcccag atgacattac cctgttatcc ctagatacat taccctgtta
     6301 tcccagatga cataccctgt tatccctaga tgacattacc ctgttatccc agataaactc
     6361 aatgatgatg atgatgatgg tcgagactca gcggccgcgg tgccagggcg tgcccttggg
     6421 ctccccgggc gcgac
//
