LOCUS       Exported                8675 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Retroviral vector contains CAG promoter , CMV enhancer and a large 
            synthetic intron that used to allow the ubiquitous expression of 
            GFP-fused Cre recombinase .
ACCESSION   .
VERSION     .
KEYWORDS    CAG-GFP/cre
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8675)
  AUTHORS   Tashiro A, Zhao C, Gage FH
  TITLE     Retrovirus-mediated single-cell gene knockout technique in adult 
            newborn neurons in vivo.
  JOURNAL   Nat Protoc. 2006;1(6):3049-55.
  PUBMED    17406567
REFERENCE   2  (bases 1 to 8675)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8675
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        135..514
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        515..718
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     LTR             719..894
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    957..1156
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             1357..1773
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1703..1725
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1741..1757
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     enhancer        1787..2166
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2169..2444
                     /label=chicken beta-actin promoter
     intron          2445..3453
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3377..3399
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      3576..3585
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             3582..4298
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             3582..4298
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3627..3648)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3888..3907)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4235..4256
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             4305..4334
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             4341..4361
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4362..5390
                     /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    5406..5994
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5459..5479)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5877..5888)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             6067..6660
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(6861..6878)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7032..7620)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7112..7131)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7798..8658)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     8421..8440
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 gatccggcca ttagccatat tattcattgg ttatatagca taaatcaata ttggctattg
       61 gccattgcat acgttgtatc catatcataa tatgtacatt tatattggct catgtccaac
      121 attaccgcca tgttgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc
      181 attagttcat agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc
      241 tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt
      301 aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca
      361 cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg
      421 taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca
      481 gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc agtacatcaa
      541 tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca ttgacgtcaa
      601 tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc
      661 cccattgacg caaatgggcg gtaggcatgt acggtgggag gtctatataa gcagagctca
      721 ataaaagagc ccacaacccc tcactcgggg cgccagtcct ccgattgact gagtcgcccg
      781 ggtacccgta ttcccaataa agcctcttgc tgtttgcatc cgaatcgtgg tctcgctgtt
      841 ccttgggagg gtctcctctg agtgattgac tacccacgac gggggtcttt catttggggg
      901 ctcgtccggg atttggagac ccctgcccag ggaccaccga cccaccaccg ggaggtaagc
      961 tggccagcaa cttatctgtg tctgtccgat tgtctagtgt ctatgtttga tgttatgcgc
     1021 ctgcgtctgt actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac
     1081 gagttctgaa cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt
     1141 tgtggcccga cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt
     1201 ctggtaggag acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt
     1261 tggaaccgaa gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc
     1321 tgtctgactg tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta
     1381 agtttgacct taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat
     1441 gtcaagaaga gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga
     1501 tggccgcgag acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt
     1561 tcacctggcc cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg
     1621 gcttttgacc cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt
     1681 cctccatccg ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc
     1741 ctttatccag ccctcactcc ttctctaggc gccggaattc gatgtcgaca ttgattattg
     1801 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     1861 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     1921 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     1981 caatgggtgg actatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     2041 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     2101 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     2161 accatgggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc
     2221 acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg
     2281 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
     2341 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
     2401 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgttgc
     2461 cttcgccccg tgccccgctc cgcgccgcct cgcgccgccc gccccggctc tgactgaccg
     2521 cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt aattagcgct
     2581 tggtttaatg acggctcgtt tcttttctgt ggctgcgtga aagccttaaa gggctccggg
     2641 agggcccttt gtgcgggggg gagcggctcg gggggtgcgt gcgtgtgtgt gtgcgtgggg
     2701 agcgccgcgt gcggcccgcg ctgcccggcg gctgtgagcg ctgcgggcgc ggcgcggggc
     2761 tttgtgcgct ccgcgtgtgc gcgaggggag cgcggccggg ggcggtgccc cgcggtgcgg
     2821 gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt gcgtgggggg gtgagcaggg
     2881 ggtgtgggcg cggcggtcgg gctgtaaccc ccccctgcac ccccctcccc gagttgctga
     2941 gcacggcccg gcttcgggtg cggggctccg tgcggggcgt ggcgcggggc tcgccgtgcc
     3001 gggcgggggg tggcggcagg tgggggtgcc gggcggggcg gggccgcctc gggccgggga
     3061 gggctcgggg gaggggcgcg gcggccccgg agcgccggcg gctgtcgagg cgcggcgagc
     3121 cgcagccatt gccttttatg gtaatcgtgc gagagggcgc agggacttcc tttgtcccaa
     3181 atctggcgga gccgaaatct gggaggcgcc gccgcacccc ctctagcggg cgcgggcgaa
     3241 gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg
     3301 ccgtcccctt ctccatctcc agcctcgggg ctgccgcagg gggacggctg ccttcggggg
     3361 ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag agcctctgct
     3421 aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct ggttgttgtg
     3481 ctgtctcatc attttggcaa agaattcgct agcggatccg gccgcctcgg ccgctagtgg
     3541 atcccccggg ctgcaggaat tcgatgctac cggtcgccac catggtgagc aagggcgagg
     3601 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     3661 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     3721 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     3781 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3841 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3901 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     3961 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     4021 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     4081 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     4141 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     4201 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     4261 ccgccgggat cactctcggc atggacgagc tgtacaagtc cggagagcaa aagctgattt
     4321 ctgaggagga tctgggagga cccaagaaga agaggaaggt gtccaattta ctgaccgtac
     4381 accaaaattt gcctgcatta ccggtcgatg caacgagtga tgaggttcgc aagaacctga
     4441 tggacatgtt cagggatcgc caggcgtttt ctgagcatac ctggaaaatg cttctgtccg
     4501 tttgccggtc gtgggcggca tggtgcaagt tgaataaccg gaaatggttt cccgcagaac
     4561 ctgaagatgt tcgcgattat cttctatatc ttcaggcgcg cggtctggca gtaaaaacta
     4621 tccagcaaca tttgggccag ctaaacatgc ttcatcgtcg gtccgggctg ccacgaccaa
     4681 gtgacagcaa tgctgtttca ctggttatgc ggcggatccg aaaagaaaac gttgatgccg
     4741 gtgaacgtgc aaaacaggct ctagcgttcg aacgcactga tttcgaccag gttcgttcac
     4801 tcatggaaaa tagcgatcgc tgccaggata tacgtaatct ggcatttctg gggattgctt
     4861 ataacaccct gttacgtata gccgaaattg ccaggatcag ggttaaagat atctcacgta
     4921 ctgacggtgg gagaatgtta atccatattg gcagaacgaa aacgctggtt agcaccgcag
     4981 gtgtagagaa ggcacttagc ctgggggtaa ctaaactggt cgagcgatgg atttccgtct
     5041 ctggtgtagc tgatgatccg aataactacc tgttttgccg ggtcagaaaa aatggtgttg
     5101 ccgcgccatc tgccaccagc cagctatcaa ctcgcgccct ggaagggatt tttgaagcaa
     5161 ctcatcgatt gatttacggc gctaaggatg actctggtca gagatacctg gcctggtctg
     5221 gacacagtgc ccgtgtcgga gccgcgcgag atatggcccg cgctggagtt tcaataccgg
     5281 agatcatgca agctggtggc tggaccaatg taaatattgt catgaactat atccgtaacc
     5341 tggatagtga aacaggggca atggtgcgcc tgctggaaga tggcgattag ctcgaggttt
     5401 aaaccaatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg
     5461 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt
     5521 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg
     5581 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc
     5641 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc
     5701 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc
     5761 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc tttccatggc
     5821 tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg
     5881 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc
     5941 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcctggaa
     6001 ttcgagctcg agcttgttaa catcgataaa ataaaagatt ttatttagtc tccagaaaaa
     6061 ggggggaatg aaagacccca cctgtaggtt tggcaagcta gcttaagtaa cgccattttg
     6121 caaggcatgg aaaaatacat aactgagaat agagaagttc agatcaaggt caggaacaga
     6181 tggaacagct gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc
     6241 agggccaaga acagatggaa cagctgaata tgggccaaac aggatatctg tggtaagcag
     6301 ttcctgcccc ggctcagggc caagaacaga tggtccccag atgcggtcca gccctcagca
     6361 gtttctagag aaccatcaga tgtttccagg gtgccccaag gacctgaaat gaccctgtgc
     6421 cttatttgaa ctaaccaatc agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg
     6481 agctcaataa aagagcccac aacccctcac tcggggcgcc agtcctccga ttgactgagt
     6541 cgcccgggta cccgtgtatc caataaaccc tcttgcagtt gcatccgact tgtggtctcg
     6601 ctgttccttg ggagggtctc ctctgagtga ttgactaccc gtcagcgggg gtctttcatt
     6661 tccgacttgt ggtctcgctg ccttgggagg gtctcctctg agtgattgac tacccgtcag
     6721 cgggggtctt cacatgcagc atgtatcaaa attaatttgg ttttttttct taagtattta
     6781 cattaaatgg ccatagttgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6841 tattggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     6901 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     6961 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     7021 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     7081 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     7141 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     7201 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     7261 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     7321 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     7381 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     7441 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg
     7501 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     7561 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     7621 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     7681 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     7741 tgaagtttgc ggccgcaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     7801 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     7861 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     7921 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     7981 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     8041 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     8101 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     8161 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     8221 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     8281 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     8341 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     8401 ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     8461 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     8521 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     8581 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     8641 gaaatgttga atactcatac tcttcctttt tcaat
//
