LOCUS       V044603                 7277 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V044603
VERSION     V044603
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     enhancer        202..581
                     /note="human cytomegalovirus immediate early enhancer"
                     /label="CMV enhancer"
     promoter        582..785
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     promoter        830..848
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label="T7 promoter"
     CDS             908..1954
                     /label="Atf4(NM_009716)"
                     /note="Atf4(NM_009716)"
                     /gene="Atf4"
     CDS             1961..2026
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     CDS             2027..2080
                     /codon_start=1
                     /note="Eukaryotic ribosomes fail to insert a peptide bond
                     between the Gly and Pro residues, yielding separate
                     polypeptides."
                     /product="2A peptide from&nbsp;Thosea asigna virus capsid
                     protein"
                     /protein_id=""
                     /transl_table=1
                     /translation="EGRGSLLTCGDVEENPGP"
                     /label="T2A"
     CDS             2081..2800
                     /codon_start=1
                     /note="mammalian codon-optimized"
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /transl_table=1
                     /translation="M,V,SKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL
                     TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFK
                     DDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNG
                     IKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMV
                     LLEFVTAAGITLGMDELYK*"
                     /label="EGFP"
     polyA_signal    2844..3068
                     /note="bovine growth hormone polyadenylation signal"
                     /label="bGH poly(A) signal"
     rep_origin      3114..3542
                     /direction=RIGHT
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
                     /label="f1 ori"
     promoter        3556..3885
                     /note="SV40 enhancer and early promoter"
                     /label="SV40 promoter"
     CDS             3952..4746
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /note="confers resistance to neomycin, kanamycin, and G418
                     (Geneticin®)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /transl_table=1
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF*"
                     /label="NeoR/KanR"
     polyA_signal    4920..5041
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     primer_bind     complement(5090..5106)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    5114..5130
                     /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-β-D-thiogalactopyranoside (IPTG)."
                     /label="lac operator"
     promoter        complement(5138..5168)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    5183..5204
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(5492..6077)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(6248..7108)
                     /codon_start=1
                     /gene="bla"
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /product="β-lactamase"
                     /transl_table=1
                     /translation="MSIQHFRVALIPFFAAFCLPVFA,HPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     SPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     WEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     ALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     SLIKHW*"
                     /label="AmpR"
     promoter        complement(7109..7213)
                     /gene="bla"
                     /label="AmpR promoter"
ORIGIN
        1 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
       61 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
      121 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
      181 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
      241 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      301 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      361 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      421 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      481 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      541 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      601 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      661 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      721 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      781 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
      841 actataggga gacccaagct ggctagcgtt taaacttaag cttggtaccg agctcggatc
      901 cgccaccatg accgagatga gcttcctgaa cagcgaagtg ttggcggggg acttgatgtc
      961 ccccttcgac cagtcgggtt tgggggctga agaaagccta ggtctcttag atgactatct
     1021 ggaggtggcc aagcacttga aacctcatgg gttctccagc gacaaggcgg gctcctcgga
     1081 atggccggct atggatgatg gcttggccag tgcctcagac accggcaagg aggatgcctt
     1141 ttccgggaca gattggatgt tggagaaaat ggatctgaaa gagtttgact tcgatgctct
     1201 gtttcgaatg gatgacctgg aaaccatgcc agatgagctc ttgaccacgt tggatgacac
     1261 atgtgatctt tttgcccctc tagtccaaga gactaataag gagccccctc agacagtgaa
     1321 cccaattggc catctcccag aaagtttaat aaaagtcgac caggttgccc cctttacatt
     1381 cttgcagcct ttcccctgtt ccccaggggt tctgtcttcc actccagagc attcctttag
     1441 tttagagcta ggcagtgaag ttgatatctc tgaaggagac aggaagcctg actctgctgc
     1501 ttacattact ctaatccctc catgtgtaaa ggaggaagac actccctctg acaatgacag
     1561 tggcatctgt atgagcccag agtcctacct gggctctccc cagcatagcc cctccacctc
     1621 cagggcccca ccagacaatc tgccttctcc aggtggttcc cgtgggtctc ctcggcccaa
     1681 accttatgac ccacctggag ttagtttgac agctaaagtg aagactgaga aattggataa
     1741 gaagctgaaa aagatggagc aaaacaagac agcagccact aggtaccgcc agaagaagcg
     1801 ggctgagcag gaggccctca ctggcgagtg taaggagcta gaaaaaaaga atgaggctct
     1861 gaaagagaag gcagattctc tggccaagga gatccagtat ctgaaagacc tgatagaaga
     1921 ggtccgtaag gcaaggggga agaagagagt tccgctcgag gactacaaag accatgacgg
     1981 tgattataaa gatcatgaca tcgactacaa ggatgacgat gacaaggagg gcagaggaag
     2041 tcttctaaca tgcggtgacg tggaggagaa tcccggccct atggtgagca agggcgagga
     2101 gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa
     2161 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt
     2221 catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta
     2281 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc
     2341 cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta
     2401 caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa
     2461 gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa
     2521 cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa
     2581 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
     2641 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc
     2701 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc
     2761 cgccgggatc actctcggca tggacgagct gtacaagtaa ctcgagtcta gagggcccgt
     2821 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     2881 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     2941 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     3001 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3061 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3121 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3181 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3241 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3301 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3361 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3421 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3481 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3541 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3601 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3661 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     3721 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     3781 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     3841 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     3901 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     3961 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4021 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4081 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     4141 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4201 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4261 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4321 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4381 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4441 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     4501 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4561 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4621 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4681 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     4741 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     4801 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     4861 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     4921 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4981 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5041 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     5101 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5161 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5221 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5281 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5341 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5401 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5461 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5521 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5581 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5641 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5701 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5761 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5821 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5881 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5941 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6001 ccggcaaaca aaccaccgct ggtagcggtt tttttgtttg caagcagcag attacgcgca
     6061 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     6121 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6181 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6241 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6301 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6361 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6421 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6481 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6541 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6601 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6661 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6721 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6781 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6841 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6901 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6961 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7021 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7081 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7141 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7201 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7261 tcccgatccc ctatggt
//
