LOCUS       Exported                7081 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    pCVL SFFV HA.NLS.I-AniIY2K227M(reo).T2A.TagBFP
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7081)
  AUTHORS   Certo MT, Ryu BY, Annis JE, Garibov M, Jarjour J, Rawlings DJ, 
            Scharenberg AM
  TITLE     Tracking genome engineering outcome at individual DNA breakpoints.
  JOURNAL   Nat Methods. 2011 Jul 10;8(8):671-6. doi: 10.1038/nmeth.1648.
  PUBMED    21743461
REFERENCE   2  (bases 1 to 7081)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7081
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        9..235
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             236..416
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    463..588
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1080..1313
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1498..1542
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1809..1926
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1955..2356
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2366..2375
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             2378..2404
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2408..2428
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3200..3253
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3254..3955
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     protein_bind    4620..4653
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(4858..4875)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4864..5088
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5161..5178)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5332..5920)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5412..5431)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6091..6951)
                     /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     6714..6733
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6952..7056)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa cgatgagtta
       61 gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg aagtaaggtg
      121 gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg gacgaaccac
      181 tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca taaacgggtc
      241 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      301 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      361 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      421 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
      481 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
      541 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
      601 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
      661 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
      721 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
      781 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
      841 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
      901 aaaagtaaga ccaccgcaca gcaagcggcc ctgatcttca gacctggagg aggagatatg
      961 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1021 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1081 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1141 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1201 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1261 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1321 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1381 aatgaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     1441 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     1501 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     1561 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     1621 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     1681 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     1741 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat
     1801 cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     1861 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     1921 aattttatcg attacgcgtc acgtgctagc tgcagtaacg ccattttgca aggcatggaa
     1981 aaataccaaa ccaagaatag agaagttcag atcaagggcg ggtacatgaa aatagctaac
     2041 gttgggccaa acaggatatc tgcggtgagc agtttcggcc ccggcccggg gccaagaaca
     2101 gatggtcacc gcagtttcgg ccccggcccg aggccaagaa cagatggtcc ccagatatgg
     2161 cccaaccctc agcagtttct taagacccat cagatgtttc caggctcccc caaggacctg
     2221 aaatgaccct gcgccttatt tgaattaacc aatcagcctg cttctcgctt ctgttcgcgc
     2281 gcttctgctt cccgagctct ataaaagagc tcacaacccc tcactcggcg cgccagtcct
     2341 ccgacagact gagtcgcccg ctcgagccac catgggatat ccatacgatg tcccagatta
     2401 tgcgccacct aagaagaaac gcaaagtccc cggggatctt acgtacgcgt atttagttgg
     2461 tctctacgaa ggggatggat actttagtat caccaagaaa ggcaagtact tgacttatga
     2521 attaggtatt gagctgagca tcaaagacgt ccaattgatt tacaagatca agaaaatcct
     2581 aggtattggc atcgtaagct tcaggaagag aaacgagatt gaaatggttg cattgaggat
     2641 ccgtgataag aatcatctaa aatctaagat attgcctata tttgagaagt atccaatgtt
     2701 ttccaacaaa cagtacgact atttaagatt caggaatgca ttgttatctg gcattatata
     2761 cctagaagac ttgcctgatt acactagaag tgacgaacca ttgaattcta tagaatccat
     2821 tatcaacaca tcatacttct ccgcctggct agttggattt atagaagctg agggctgttt
     2881 cagtgtgtac aagctgaaca aagacgatga ctacttgatt gcttcattcg acattgccca
     2941 aagagatggt gatatcttga tttcagcaat taggaagtac ttaagtttca ctactaaggt
     3001 ttacctagac aagactaatt gtagcaaatt gaaggtcact agtgttagat ccgtcgagaa
     3061 catcattaag tttctgcaga atgctcctgt caaattgtta ggcaacatga aactgcaata
     3121 caagttgtgg ttgaaacaac taaggaagat ttctaggtat tccgagaaga tcaagattcc
     3181 atcaaactac ggatccggtg agggcagagg aagtcttcta acatgcggtg acgtggagga
     3241 gaatccgggc cccatgagcg agctgattaa ggagaacatg cacatgaagc tgtacatgga
     3301 gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca agccctacga
     3361 gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct tcgccttcga
     3421 catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca cccagggcat
     3481 ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag tcaccacata
     3541 cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg gctgcctcat
     3601 ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga tgcagaagaa
     3661 aacactcggc tgggaggcct tcaccgagac gctgtacccc gctgacggcg gcctggaagg
     3721 cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa acatcaagac
     3781 cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct actatgtgga
     3841 ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc agcacgaggt
     3901 ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctta attgattcta
     3961 gagtcgaccg agcatcttac cgccatttat acccatattt gttctgtttt tcttgatttg
     4021 ggtatacatt taaatgttaa tagaacaaaa tggtggggca atcatttaca tttttaggga
     4081 tatgtaatta ctagttcagg tgtattgcca caagacaaac atgttaagaa actttcccgt
     4141 tatttacgct ctgttcctgt taatcaacct ctggattaca aaatttgtga aagattgact
     4201 gatattctta actatgttgc tccttttacg ctgtgtggat atgctgcttt atagcctctg
     4261 tatctagcta ttgcttcccg tacggctttc gttttctcct ccttgtataa atcctggttg
     4321 ctgtctcttt tagaggagtt gtggcccgtt gtccgtcaac gtggcgtggt gtgctctgtg
     4381 tttgctgacg caacccccac tggctggggc attgccacca cctgtcaact cctttctggg
     4441 actttcgctt tccccctccc gatcgccacg gcagaactca tcgccgcctg ccttgcccgc
     4501 tgctggacag gggctaggtt gctgggcact gataattccg tggtgttgtc atcggtacct
     4561 ttttaaaaga aaagggggga ctggaagggc taattcactc ccaacgaaga caagatatca
     4621 taacttcgta tagcatacat tatacgaagt tataatttat ttgtgaaatt tgtgatgcta
     4681 ttgctttatt tgtaaccata tgtttatttg tgaaatttgt gatgctattg ctttatttgt
     4741 aaccattgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc
     4801 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct cgaccagcct
     4861 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga
     4921 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt
     4981 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg
     5041 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggcc tgcagctgca
     5101 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     5161 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     5221 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     5281 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5341 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5401 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5461 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5521 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5581 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5641 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5701 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5761 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5821 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5881 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5941 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     6001 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     6061 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     6121 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     6181 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     6241 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6301 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6361 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6421 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6481 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6541 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6601 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6661 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6721 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6781 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6841 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6901 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6961 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     7021 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     7081 t
//
