LOCUS       sequence-384303-        7881 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses Cre recombinase and puromycin N-acetyltransferase driven 
            by the mammalian EF1a promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7881)
  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   Nat Protoc. 2008;3(10):1550-8. doi: 10.1038/nprot.2008.146.
  PUBMED    18802436
REFERENCE   2  (bases 1 to 7881)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Protoc."; date: "2008"; volume: "3(10):1550-8. doi"; pages: " 
            10.1038/nprot.2008.146"
COMMENT     Sequence Label: pEFBOS_CreIRESpuro
FEATURES             Location/Qualifiers
     source          1..7881
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        253..463
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     promoter        275..471
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      322..457
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        558..1736
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          789..1727
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1684..1704
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1759..1779
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1780..2808
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     misc_feature    join(2842..3416,3417..3419,3420..3428)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3009..3026)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3236..3255
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3441..4037
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     3934..3954
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4112..4129)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4118..4342
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5010..5027)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5010..5026)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5019..5041)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5239..5694
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5326..5345)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5536..5557
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5711..5730)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5830..5852
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5890..5908)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5976..6080
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6081..6149,6150..6941)
                     /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     complement(6299..6318)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7112..7700
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7601..7620
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7854..7871
                     /label=L4440
                     /note="L4440 vector, forward primer"
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 gccaagctta
      241 attccctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
      301 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
      361 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
      421 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctttgc
      481 aaagatggat aaagttttaa acagagagga atctttgcag ctaatggacc ttctaggtct
      541 tgaaaggagt gggaattggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag
      601 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg
      661 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag
      721 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca
      781 gaacacaggt aagtgccgtg tgtggttccc gcgggcctgg cctctttacg ggttatggcc
      841 cttgcgtgcc ttgaattact tccacctggc tgcagtacgt gattcttgat cccgagcttc
      901 gggttggaag tgggtgggag agttcgaggc cttgcgctta aggagcccct tcgcctcgtg
      961 cttgagttga ggcctggcct gggcgctggg gccgccgcgt gcgaatctgg tggcaccttc
     1021 gcgcctgtct cgctgctttc gataagtctc tagccattta aaatttttga tgacctgctg
     1081 cgacgctttt tttctggcaa gatagtcttg taaatgcggg ccaagatctg cacactggta
     1141 tttcggtttt tggggccgcg ggcggcgacg gggcccgtgc gtcccagcgc acatgttcgg
     1201 cgaggcgggg cctgcgagcg cggccaccga gaatcggacg ggggtagtct caagctggcc
     1261 ggcctgctct ggtgcctggc ctcgcgccgc cgtgtatcgc cccgccctgg gcggcaaggc
     1321 tggcccggtc ggcaccagtt gcgtgagcgg aaagatggcc gcttcccggc cctgctgcag
     1381 ggagctcaaa atggaggacg cggcgctcgg gagagcgggc gggtgagtca cccacacaaa
     1441 ggaaaagggc ctttccgtcc tcagccgtcg cttcatgtga ctccacggag taccgggcgc
     1501 cgtccaggca cctcgattag ttctcgagct tttggagtac gtcgtcttta ggttgggggg
     1561 aggggtttta tgcgatggag tttccccaca ctgagtgggt ggagactgaa gttaggccag
     1621 cttggcactt gatgtaattc tccttggaat ttgccctttt tgagtttgga tcttggttca
     1681 ttctcaagcc tcagacagtg gttcaaagtt tttttcttcc atttcaggtg tcgtgaggaa
     1741 ttctctaggt cgaccatgcc caagaagaag aggaaggtgt ccaatttact gaccgtacac
     1801 caaaatttgc ctgcattacc ggtcgatgca acgagtgatg aggttcgcaa gaacctgatg
     1861 gacatgttca gggatcgcca ggcgttttct gagcatacct ggaaaatgct tctgtccgtt
     1921 tgccggtcgt gggcggcatg gtgcaagttg aataaccgga aatggtttcc cgcagaacct
     1981 gaagatgttc gcgattatct tctatatctt caggcgcgcg gtctggcagt aaaaactatc
     2041 cagcaacatt tgggccagct aaacatgctt catcgtcggt ccgggctgcc acgaccaagt
     2101 gacagcaatg ctgtttcact ggttatgcgg cggatccgaa aagaaaacgt tgatgccggt
     2161 gaacgtgcaa aacaggctct agcgttcgaa cgcactgatt tcgaccaggt tcgttcactc
     2221 atggaaaata gcgatcgctg ccaggatata cgtaatctgg catttctggg gattgcttat
     2281 aacaccctgt tacgtatagc cgaaattgcc aggatcaggg ttaaagatat ctcacgtact
     2341 gacggtggga gaatgttaat ccatattggc agaacgaaaa cgctggttag caccgcaggt
     2401 gtagagaagg cacttagcct gggggtaact aaactggtcg agcgatggat ttccgtctct
     2461 ggtgtagctg atgatccgaa taactacctg ttttgccggg tcagaaaaaa tggtgttgcc
     2521 gcgccatctg ccaccagcca gctatcaact cgcgccctgg aagggatttt tgaagcaact
     2581 catcgattga tttacggcgc taaggatgac tctggtcaga gatacctggc ctggtctgga
     2641 cacagtgccc gtgtcggagc cgcgcgagat atggcccgcg ctggagtttc aataccggag
     2701 atcatgcaag ctggtggctg gaccaatgta aatattgtca tgaactatat ccgtaacctg
     2761 gatagtgaaa caggggcaat ggtgcgcctg ctggaagatg gcgattagcc attaacgcgg
     2821 atccctcgac cccgggatcc gcccctctcc ctcccccccc cctaacgtta ctggccgaag
     2881 ccgcttggaa taaggccggt gtgcgtttgt ctatatgtta ttttccacca tattgccgtc
     2941 ttttggcaat gtgagggccc ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg
     3001 tctttcccct ctcgccaaag gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc
     3061 tctggaagct tcttgaagac aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc
     3121 cccacctggc gacaggtgcc tctgcggcca aaagccacgt gtataagata cacctgcaaa
     3181 ggcggcacaa ccccagtgcc acgttgtgag ttggatagtt gtggaaagag tcaaatggct
     3241 ctcctcaagc gtattcaaca aggggctgaa ggatgcccag aaggtacccc attgtatggg
     3301 atctgatctg gggcctcggt acacatgctt tacatgtgtt tagtcgaggt taaaaaaacg
     3361 tctaggcccc ccgaaccacg gggacgtggt tttcctttga aaaacacgat gataatatgg
     3421 ccacaaccat ggcgcgcctt accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     3481 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     3541 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     3601 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     3661 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     3721 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     3781 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     3841 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     3901 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     3961 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     4021 gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac
     4081 gaccccatgc atcgtagagc tcgctgatca gcctcgactg tgccttctag ttgccagcca
     4141 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     4201 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     4261 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     4321 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctcgatggcg
     4381 cgttctagag tgagggtccc cacctgggac ccttgagagt atcaggtctc ccacgtggga
     4441 gacaagaaat ccctgtttaa tatttaaaca gcagtgttcc ccatctgggt ccttgcaccc
     4501 ctcactctgg cctcagccga ctgcacagcg gcccctgcat ccccttggct gtgaggcccc
     4561 tggacaagca gaggtggcca gagctgggag gcatggccct ggggtcccac gaatttgctg
     4621 gggaatctcg tttttcttct taagactttt gggacatggt ttgactcccg aacatcaccg
     4681 acgtgtctcc tgtttttctg ggtggcctcg ggacacctgc cctgccccca cgagggtcag
     4741 gactgtgact ctttttaggg ccaggcaggt gcctggacat ttgccttgct ggacggggac
     4801 tggggatgtg ggagggagca gacaggagga atcatgtcag gcctgtgtgt gaaaggaagc
     4861 tccactgtca ccctccacct cttcaccccc cactcaccag tgtcccctcc actgtcacat
     4921 tgtaactgaa cttcaggata awaaagtgtt tgccyccaaa aaaaaaaaaa aaaaaaaaaa
     4981 aaaaaaaaaa aaaaaaaaaa accgaattca ctggccgtcg ttttacaacg tcgtgactgg
     5041 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     5101 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     5161 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5221 cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg
     5281 ttacgcgcag cgtgaccgct acacttgcca gcgccttagc gcccgctcct ttcgctttct
     5341 tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc
     5401 ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg
     5461 atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt
     5521 ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac tctatctcgg
     5581 gctattcttt tgatttataa gggattttgc cgatttcggt ctattggtta aaaaatgagc
     5641 tgatttaaca aaaatttaac gcgaatttta acaaaatatt aacgtttaca attttatggt
     5701 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     5761 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     5821 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5881 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     5941 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     6001 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     6061 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     6121 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     6181 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     6241 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     6301 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     6361 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     6421 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     6481 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     6541 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     6601 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     6661 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     6721 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     6781 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     6841 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     6901 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     6961 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     7021 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     7081 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     7141 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     7201 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     7261 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     7321 tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg
     7381 ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
     7441 cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg
     7501 agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
     7561 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
     7621 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
     7681 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
     7741 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
     7801 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
     7861 cagtgagcga ggaagcggaa g
//
