LOCUS       sequence-37996-e        7998 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7998)
  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 7998)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: "10.1038/nmeth"; 
            journalName: "Nat Methods"; date: "2011-07-10- 10"; volume: "8"; 
            issue: "8"; pages: "671-6"
COMMENT     Sequence Label: pCVL.SFFV.d14GFP.EF1a.HA.NLS.Sce(opt).T2A.TagBFP
FEATURES             Location/Qualifiers
     source          1..7998
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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..2361
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2376..2385
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2382..2384,2385..2387,2388..2900)
                     /codon_start=1
                     /product="N-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN173
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIE"
     primer_bind     complement(2427..2448)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2688..2707)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3035..3056
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     promoter        3086..3297
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      3306..3315
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3321..3347
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             3321..3347
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             3357..3377
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4098..4151
                     /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             4152..4853
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    4867..5455
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4920..4940)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5338..5349)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(5537..5570)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(5775..5792)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5781..6005
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6078..6095)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6249..6837)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6329..6348)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7008..7799,7800..7868))
                     /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     7631..7650
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7869..7973)
                     /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 gatcgatcct cgagcgccac catggtgagc aagggcgagg
     2401 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     2461 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     2521 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     2581 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     2641 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     2701 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     2761 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     2821 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     2881 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     2941 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     3001 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgtgag
     3061 gatccaggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
     3121 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
     3181 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
     3241 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggat
     3301 ctgccgccac catgggcgta tacccctacg acgtgcccga ctacgccccc gggcccccta
     3361 agaaaaagag gaaggtgaag aacatcaaga agaaccaggt catgaacctg ggccccaaca
     3421 gcaagctgct gaaggagtac aagagccagc tgatcgagct gaacatcgag cagttcgagg
     3481 ccggcatcgg cctgatcctg ggcgacgcct acatcaggag cagggacgag ggcaagacct
     3541 actgcatgca gttcgagtgg aagaacaagg cctacatgga ccacgtgtgc ctgctgtacg
     3601 accagtgggt gctgagcccc ccccacaaga aggagagggt gaaccacctg ggcaacctgg
     3661 tcatcacctg gggcgcccag accttcaagc accaggcctt caacaagctg gccaacctgt
     3721 tcatcgtgaa caacaagaag accatcccca acaacctggt ggagaactac ctgaccccca
     3781 tgagcctggc ctactggttc atggacgacg gcggcaagtg ggactacaac aagaacagca
     3841 ccaacaagag catcgtgctg aacacccaga gcttcacctt cgaggaggtg gagtacctgg
     3901 tgaagggcct gaggaacaag ttccagctga actgctacgt gaagatcaac aagaacaagc
     3961 ccatcatcta catcgacagc atgagctacc tgatcttcta caacctgatc aagccctacc
     4021 tgatccccca gatgatgtac aagctgccca acaccatcag cagcgagacc ttcctgaagg
     4081 gcggcggcgg atccggtgag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga
     4141 atccgggccc catgagcgag ctgattaagg agaacatgca catgaagctg tacatggagg
     4201 gcaccgtgga caaccatcac ttcaagtgca catccgaggg cgaaggcaag ccctacgagg
     4261 gcacccagac catgagaatc aaggtggtcg agggcggccc tctccccttc gccttcgaca
     4321 tcctggctac tagcttcctc tacggcagca agaccttcat caaccacacc cagggcatcc
     4381 ccgacttctt caagcagtcc ttccctgagg gcttcacatg ggagagagtc accacatacg
     4441 aagacggggg cgtgctgacc gctacccagg acaccagcct ccaggacggc tgcctcatct
     4501 acaacgtcaa gatcagaggg gtgaacttca catccaacgg ccctgtgatg cagaagaaaa
     4561 cactcggctg ggaggccttc accgagacgc tgtaccccgc tgacggcggc ctggaaggca
     4621 gaaacgacat ggccctgaag ctcgtgggcg ggagccatct gatcgcaaac atcaagacca
     4681 catatagatc caagaaaccc gctaagaacc tcaagatgcc tggcgtctac tatgtggact
     4741 acagactgga aagaatcaag gaggccaaca acgagaccta cgtcgagcag cacgaggtgg
     4801 cagtggccag atactgcgac ctccctagca aactggggca caagcttaat tgattctaga
     4861 gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat
     4921 gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct
     4981 tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag
     5041 gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc
     5101 cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc
     5161 ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct
     5221 cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga agctgacgtc ctttccatgg
     5281 ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg
     5341 gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg
     5401 cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcctgga
     5461 attcgagctc ggtacctttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     5521 acgaagacaa gatatcataa cttcgtatag catacattat acgaagttat aatttatttg
     5581 tgaaatttgt gatgctattg ctttatttgt aaccatatgt ttatttgtga aatttgtgat
     5641 gctattgctt tatttgtaac cattgctttt tgcttgtact gggtctctct ggttagacca
     5701 gatctgagcc tgggagctct ctggctaact agggaaccca ctgcttaagc ctcaataaag
     5761 cttgcctcga ccagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     5821 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     5881 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     5941 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     6001 tatggcctgc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     6061 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     6121 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     6181 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     6241 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     6301 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     6361 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     6421 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     6481 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     6541 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     6601 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     6661 tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
     6721 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     6781 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     6841 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     6901 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     6961 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     7021 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     7081 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     7141 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     7201 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     7261 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     7321 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     7381 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     7441 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     7501 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     7561 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     7621 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     7681 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     7741 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     7801 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     7861 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     7921 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     7981 ccccgaaaag tgccacct
//
