LOCUS       40924_17089        8102 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pEFBOS-creIRESPuro, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8102)
  AUTHORS   Davis RP, Ng ES, Costa M, Mossman AK, Sourris K, Elefanty AG, 
            Stanley EG.
  TITLE     Targeting a GFP reporter gene to the MIXL1 locus of human embryonic 
            stem cells identifies human primitive streak-like cells and enables 
            isolation of primitive hematopoietic precursors
  JOURNAL   Blood 111 (4), 1876-1884 (2008)
  PUBMED    18032708
REFERENCE   2  (bases 1 to 8102)
  AUTHORS   Davis RP, Costa M, Grandela C, Holland AM, Hatzistavrou T, Micallef 
            SJ, Li X, Goulburn AL, Azzola L, Elefanty AG, Stanley EG.
  TITLE     A protocol for removal of antibiotic resistance cassettes from human
            embryonic stem cells genetically modified by homologous 
            recombination or transgenesis
  JOURNAL   Unpublished
REFERENCE   3  (bases 1 to 8102)
  AUTHORS   Davis RP, Stanley EG, Elefanty AG.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-MAY-2008) MISCL, Monash University, Wellington, 
            Clayton, Victoria 3800, Australia
REFERENCE   4  (bases 1 to 8102)
  TITLE     Direct Submission
REFERENCE   5  (bases 1 to 8102)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Blood"; 
            date: "2008"; volume: "111"; issue: "4"; pages: "1876-1884"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 3; type: "Journal Article"; journalName: "Submitted 
            (02-MAY-2008) MISCL, Monash University, Wellington, Clayton, 
            Victoria 3800, Australia"
COMMENT     SGRef: number: 4; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8102
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        143..173
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(333..543)
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        562..1743
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation
                     factor EF-1-alpha"
     CDS             1766..1786
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1784..2812
                     /codon_start=1
                     /label=Cre
                     /note="site-specific recombinase"
                     /translation="VSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     misc_feature    2849..3435
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     CDS             3445..4041
                     /codon_start=1
                     /label=PuroR
                     /note="puromycin N-acetyltransferase"
                     /translation="LTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     polyA_signal    4125..4349
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     3'UTR           4522..5222
                     /label=bovine growth hormone 3' UTR
                     /note="bovine growth hormone 3' UTR"
     primer_bind     complement(5231..5247)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      5460..5915
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6197..6301
                     /label=AmpR promoter
     CDS             6302..7159
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7333..7921
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttc
      241 caggcactcc tttcaagacc tagaaggtcc attagctgca aagattcctc tctgtttaaa
      301 actttatcca tctttgcaaa gctttttgca aaagcctagg cctccaaaaa agcctcctca
      361 ctacttctgg aatagctcag aggccgaggc ggcctcggcc tctgcataaa taaaaaaaat
      421 tagtcagcca tggggcggag aatgggcgga actgggcgga gttaggggcg ggatgggcgg
      481 agttaggggc gggactatgg ttgctgacta attgagatgc atgctttgca tacttctgcc
      541 tgctggggag cctggcgtga ggctccggtg cccgtcagtg ggcagagcgc acatcgccca
      601 cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc
      661 gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg
      721 gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg
      781 ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg
      841 gcccttgcgt gccttgaatt acttccacgc ccctggctgc agtacgtgat tcttgatccc
      901 gagcttcggg ttggaagtgg gtgggagagt tcgaggcctt gcgcttaagg agccccttcg
      961 cctcgtgctt gagttgaggc ctggcctggg cgctggggcc gccgcgtgcg aatctggtgg
     1021 caccttcgcg cctgtctcgc tgctttcgat aagtctctag ccatttaaaa tttttgatga
     1081 cctgctgcga cgcttttttt ctggcaagat agtcttgtaa atgcgggcca agatctgcac
     1141 actggtattt cggtttttgg ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca
     1201 tgttcggcga ggcggggcct gcgagcgcgg ccaccgagaa tcggacgggg gtagtctcaa
     1261 gctggccggc ctgctctggt gcctggcctc gcgccgccgt gtatcgcccc gccctgggcg
     1321 gcaaggctgg cccggtcggc accagttgcg tgagcggaaa gatggccgct tcccggccct
     1381 gctgcaggga gctcaaaatg gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc
     1441 acacaaagga aaagggcctt tccgtcctca gccgtcgctt catgtgactc cacggagtac
     1501 cgggcgccgt ccaggcacct cgattagttc tcgagctttt ggagtacgtc gtctttaggt
     1561 tggggggagg ggttttatgc gatggagttt ccccacactg agtgggtgga gactgaagtt
     1621 aggccagctt ggcacttgat gtaattctcc ttggaatttg ccctttttga gtttggatct
     1681 tggttcattc tcaagcctca gacagtggtt caaagttttt ttcttccatt tcaggtgtcg
     1741 tgaggaattc tctaggtcga ccatgcccaa gaagaagagg aaggtgtcca atttactgac
     1801 cgtacaccaa aatttgcctg cattaccggt cgatgcaacg agtgatgagg ttcgcaagaa
     1861 cctgatggac atgttcaggg atcgccaggc gttttctgag catacctgga aaatgcttct
     1921 gtccgtttgc cggtcgtggg cggcatggtg caagttgaat aaccggaaat ggtttcccgc
     1981 agaacctgaa gatgttcgcg attatcttct atatcttcag gcgcgcggtc tggcagtaaa
     2041 aactatccag caacatttgg gccagctaaa catgcttcat cgtcggtccg ggctgccacg
     2101 accaagtgac agcaatgctg tttcactggt tatgcggcgg atccgaaaag aaaacgttga
     2161 tgccggtgaa cgtgcaaaac aggctctagc gttcgaacgc actgatttcg accaggttcg
     2221 ttcactcatg gaaaatagcg atcgctgcca ggatatacgt aatctggcat ttctggggat
     2281 tgcttataac accctgttac gtatagccga aattgccagg atcagggtta aagatatctc
     2341 acgtactgac ggtgggagaa tgttaatcca tattggcaga acgaaaacgc tggttagcac
     2401 cgcaggtgta gagaaggcac ttagcctggg ggtaactaaa ctggtcgagc gatggatttc
     2461 cgtctctggt gtagctgatg atccgaataa ctacctgttt tgccgggtca gaaaaaatgg
     2521 tgttgccgcg ccatctgcca ccagccagct atcaactcgc gccctggaag ggatttttga
     2581 agcaactcat cgattgattt acggcgctaa ggatgactct ggtcagagat acctggcctg
     2641 gtctggacac agtgcccgtg tcggagccgc gcgagatatg gcccgcgctg gagtttcaat
     2701 accggagatc atgcaagctg gtggctggac caatgtaaat attgtcatga actatatccg
     2761 taacctggat agtgaaacag gggcaatggt gcgcctgctg gaagatggcg attagccatt
     2821 aacgcggatc cctcgacccc gggatccgcc cctctccctc ccccccccct aacgttactg
     2881 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat
     2941 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc
     3001 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag
     3061 cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc
     3121 ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac
     3181 ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca
     3241 aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt
     3301 gtatgggatc tgatctgggg cctcggtaca catgctttac atgtgtttag tcgaggttaa
     3361 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat
     3421 aatatggcca caaccatggc gcgccttacc gagtacaagc ccacggtgcg cctcgccacc
     3481 cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc
     3541 acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct gcaagaactc
     3601 ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg
     3661 gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc
     3721 ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat ggaaggcctc
     3781 ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc
     3841 gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag
     3901 cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc cttctacgag
     3961 cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc
     4021 atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg
     4081 agcgcacgac cccatgcatc gtagagctcg ctgatcagcc tcgactgtgc cttctagttg
     4141 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     4201 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     4261 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     4321 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     4381 gatggcgcgt tctagagtga gggtccccac ctgggaccct tgagagtatc aggtctccca
     4441 cgtgggagac aagaaatccc tgtttaatat ttaaacagca gtgttcccca tctgggtcct
     4501 tgcacccctc actctggcct cagccgactg cacagcggcc cctgcatccc cttggctgtg
     4561 aggcccctgg acaagcagag gtggccagag ctgggaggca tggccctggg gtcccacgaa
     4621 tttgctgggg aatctcgttt ttcttcttaa gacttttggg acatggtttg actcccgaac
     4681 atcaccgacg tgtctcctgt ttttctgggt ggcctcggga cacctgccct gcccccacga
     4741 gggtcaggac tgtgactctt tttagggcca ggcaggtgcc tggacatttg ccttgctgga
     4801 tggggactgg ggatgtggga gggagcagac aggaggaatc atgtcaggcc tgtgtgtgaa
     4861 aggaagctcc actgtcaccc tccacctctt caccccccac tcaccagtgt cccctccact
     4921 gtcacattgt aactgaactt caggataata aagtgtttgc ctccagtcac gtccttcctc
     4981 cttcttgagt ccagctggtg cctggccagg ggctggggag gtggctgaag ggtgggagag
     5041 gccagaggga ggtcggggag gaggtctggg gaggaggtcc agggaggagg aggaaagttc
     5101 tcaagttcgt ctgacattca ttccgttagc acatatttat ctgagcacct actctgtgca
     5161 gacgctgggc taagtgctgg ggacacagca gggaacaagg cagacatgga atctgcactc
     5221 ganngaattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     5281 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     5341 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt
     5401 attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta
     5461 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5521 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5581 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5641 tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc
     5701 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     5761 actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata
     5821 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     5881 cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg
     5941 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
     6001 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     6061 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat
     6121 acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac
     6181 ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat
     6241 gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag
     6301 tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc
     6361 tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc
     6421 acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc
     6481 cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc
     6541 ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt
     6601 ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt
     6661 atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat
     6721 cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct
     6781 tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat
     6841 gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc
     6901 ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg
     6961 ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc
     7021 tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta
     7081 cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc
     7141 ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga
     7201 tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     7261 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     7321 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     7381 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     7441 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt
     7501 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     7561 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     7621 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     7681 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     7741 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     7801 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     7861 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     7921 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     7981 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     8041 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     8101 ag
//
