LOCUS       V032998                 9424 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   V032998
VERSION     V032998
KEYWORDS    .
SOURCE      .
  ORGANISM  .
            .
FEATURES             Location/Qualifiers
     LTR             234..414
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
                     /label="5' LTR (truncated)"
     misc_feature    458..583
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
                     /label="HIV-1 Ψ"
     misc_feature    1076..1309
                     /note="The Rev response element (RRE) of HIV-1 allows for
                     Rev-dependent mRNA export from the nucleus to the
                     cytoplasm."
                     /label="RRE"
     CDS             1493..1537
                     /codon_start=1
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /product="antigenic peptide corresponding to amino acids
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik
                     et al., 2013)"
                     /transl_table=1
                     /translation="KNEQELLELDKWASL"
                     /label="gp41 peptide"
     misc_feature    1804..1921
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
                     /label="cPPT/CTS"
     promoter        2011..2214
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label="CMV promoter"
     CDS             2317..3450
                     /label="METTL2A(NM_181725)"
                     /note="METTL2A(NM_181725)"
                     /gene="METTL2A"
     CDS             3457..3522
                     /codon_start=1
                     /product="three tandem FLAG® epitope tags, followed by an
                     enterokinase cleavage site"
                     /transl_table=1
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
                     /label="3xFLAG"
     promoter        3566..3777
                     /note="core&nbsp;promoter for human elongation factor
                     EF-1α"
                     /label="EF-1α core promoter"
     LTR             3790..4058
                     /note="truncated 5' long terminal repeat (LTR) from human
                     T-cell leukemia virus (HTLV) type 1"
                     /label="5' LTR (truncated)"
     regulatory      4087..4096
                     /note="vertebrate consensus sequence for strong initiation
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
                     /label="Kozak sequence"
     CDS             4117..4779
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /transl_table=1
                     /translation="PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALT
                     FSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRY
                     EAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGG
                     YYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA"
                     /label="CopGFP"
     CDS             4849..4902
                     /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             4903..5502
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /note="confers resistance to puromycin"
                     /product="puromycin N-acetyltransferase"
                     /transl_table=1
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA*"
                     /label="PuroR"
     misc_feature    5503..6091
                     /note="woodchuck hepatitis virus&nbsp;posttranscriptional
                     regulatory element"
                     /label="WPRE"
     LTR             6165..6398
                     /note="self-inactivating&nbsp;3' long terminal repeat (LTR)
                     from HIV-1"
                     /label="3' LTR (ΔU3)"
     polyA_signal    6470..6591
                     /note="SV40 polyadenylation signal"
                     /label="SV40 poly(A) signal"
     rep_origin      6631..6766
                     /note="SV40 origin of replication"
                     /label="SV40 ori"
     primer_bind     complement(6799..6815)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 rev"
     protein_bind    6823..6839
                     /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(6847..6877)
                     /note="promoter for the E. coli lac operon"
                     /label="lac promoter"
     protein_bind    6892..6913
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label="CAP binding site"
     rep_origin      complement(7201..7789)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label="ori"
     CDS             complement(7960..8820)
                     /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(8821..8925)
                     /gene="bla"
                     /label="AmpR promoter"
     primer_bind     9399..9415
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label="M13 fwd"
ORIGIN
        1 acgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca
       61 acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta
      121 cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga
      181 attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc
      241 tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta
      301 agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact
      361 ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg
      421 cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct
      481 tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt
      541 gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag
      601 aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt
      661 aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt
      721 agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg
      781 atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag
      841 gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag
      901 taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg
      961 acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag
     1021 cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag
     1081 ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc
     1141 tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga
     1201 gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc
     1261 aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg
     1321 gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata
     1381 aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa
     1441 ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga
     1501 acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa
     1561 ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat
     1621 agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt
     1681 tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg
     1741 tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt
     1801 aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     1861 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     1921 tatcgatact agtattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     1981 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     2041 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     2101 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     2161 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggttt atataagcag agctcgttta
     2221 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagattc
     2281 tagagctagc ctgatacgaa ctcggaattc gccaccatgg ccggctccta ccctgaaggt
     2341 gcacctgcag tcctcgccga taagaggcag cagttcggaa gccggttcct gagagatccg
     2401 gcgcgcgtct tccaccacaa tgcctgggac aatgtggagt ggtcggaaga gcaagccgcg
     2461 gcggcggaga gaaaagtcca ggagaacagt atccagcggg tgtgccagga gaaacaagtt
     2521 gattatgaga tcaatgccca caaatactgg aatgacttct acaaaatcca cgaaaatggg
     2581 tttttcaagg atagacattg gctttttacc gaattccctg agctggcacc tagccaaaat
     2641 caaaatcatt tgaaggactg gttcttggag aacaagagtg aagtacctga atgtagaaac
     2701 aatgaggatg gacctggttt aataatggaa gaacagcaca agtgttcttc aaagagcctt
     2761 gaacataaaa cacagacact tcctgtggag gagaatgtaa ctcagaaaat tagtgacctg
     2821 gaaatttgtg ctgatgagtt tcctggatcc tcagccacct accgaatact ggaggttggc
     2881 tgtggtgtgg gaaacacagt ctttccaatt ttacaaacga acaatgaccc aggactcttt
     2941 gtttattgct gtgatttttc ttccacagct atagaactgg tccagacaaa ttcagaatat
     3001 gatccttctc ggtgttttgc ctttgttcac gacctgtgtg atgaagagaa gagttaccca
     3061 gtgcccaagg gcagtcttga tattatcatt ctcatatttg ttctttcagc aattgttcca
     3121 gacaagatgc agaaggctat caacaggctg agcaggcttc tgaaacctgg cgggatgatg
     3181 cttctgcgag attacggccg ctatgacatg gctcagcttc ggtttaaaaa aggtcagtgt
     3241 ctatctggaa atttctacgt gagaggtgat ggaaccagag tttacttctt cacacaagag
     3301 gaactggaca cgcttttcac cactgctgga ctggaaaaag ttcagaacct ggtggatcgc
     3361 cgactgcagg tgaaccgagg aaagcaactg acaatgtacc gggtttggat tcagtgcaaa
     3421 tactgcaagc cccttctgtc cagcaccagc ggatccgact acaaagacca tgacggtgat
     3481 tataaagatc atgacatcga ctacaaggat gacgatgaca agtagcggcc gcgaaggatc
     3541 tgcgatcgct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     3601 ttggggggag gggtcggcaa ttgaacgggt gcctagagaa ggtggcgcgg ggtaaactgg
     3661 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     3721 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacagctg
     3781 aagcttcgag gggctcgcat ctctccttca cgcgcccgcc gccctacctg aggccgccat
     3841 ccacgccggt tgagtcgcgt tctgccgcct cccgcctgtg gtgcctcctg aactgcgtcc
     3901 gccgtctagg taagtttaaa gctcaggtcg agaccgggcc tttgtccggc gctcccttgg
     3961 agcctaccta gactcagccg gctctccacg ctttgcctga ccctgcttgc tcaactctac
     4021 gtctttgttt cgttttctgt tctgcgccgt tacagatcca agctgtgacc ggcgcctacg
     4081 ctagacgcca ccatggagag cgacgagagc ggcctgcccg ccatggagat cgagtgccgc
     4141 atcaccggca ccctgaacgg cgtggagttc gagctggtgg gcggcggaga gggcaccccc
     4201 aagcagggcc gcatgaccaa caagatgaag agcaccaaag gcgccctgac cttcagcccc
     4261 tacctgctga gccacgtgat gggctacggc ttctaccact tcggcaccta ccccagcggc
     4321 tacgagaacc ccttcctgca cgccatcaac aacggcggct acaccaacac ccgcatcgag
     4381 aagtacgagg acggcggcgt gctgcacgtg agcttcagct accgctacga ggccggccgc
     4441 gtgatcggcg acttcaaggt ggtgggcacc ggcttccccg aggacagcgt gatcttcacc
     4501 gacaagatca tccgcagcaa cgccaccgtg gagcacctgc accccatggg cgataacgtg
     4561 ctggtgggca gcttcgcccg caccttcagc ctgcgcgacg gcggctacta cagcttcgtg
     4621 gtggacagcc acatgcactt caagagcgcc atccacccca gcatcctgca gaacgggggc
     4681 cccatgttcg ccttccgccg cgtggaggag ctgcacagca acaccgagct gggcatcgtg
     4741 gagtaccagc acgccttcaa gacccccatc gccttcgcca gatcccgcgc tcagtcgtcc
     4801 aattctgccg tggacggcac cgccggaccc ggctccaccg gatctcgcga gggcagagga
     4861 agtcttctaa catgcggtga cgtggaggag aatcccggcc ctatgaccga gtacaagccc
     4921 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     4981 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     5041 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     5101 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     5161 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     5221 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     5281 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     5341 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     5401 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     5461 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gaaatcaacc tctggattac
     5521 aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga
     5581 tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc
     5641 tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa
     5701 cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc
     5761 acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc
     5821 atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc
     5881 gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg
     5941 attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct
     6001 tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg
     6061 agtcggatct ccctttgggc cgcctccccg cctggtacct ttaagaccaa tgacttacaa
     6121 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca
     6181 ctcccaacga agataagatc tgctttttgc ttgtactggg tctctctggt tagaccagat
     6241 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     6301 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     6361 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat
     6421 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat
     6481 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     6541 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg
     6601 gctctagcta tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     6661 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     6721 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctaga cttttgcaga
     6781 gaccaaattc gtaatcatgt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     6841 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     6901 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     6961 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     7021 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     7081 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     7141 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     7201 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     7261 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     7321 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     7381 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     7441 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     7501 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     7561 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7621 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     7681 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7741 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     7801 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7861 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     7921 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7981 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     8041 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     8101 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     8161 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     8221 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     8281 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     8341 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     8401 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     8461 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     8521 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     8581 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     8641 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     8701 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     8761 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8821 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     8881 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     8941 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     9001 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     9061 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     9121 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
     9181 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
     9241 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
     9301 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
     9361 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa
     9421 gctg
//
