LOCUS       sequence-271054-        7918 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 7918)
  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 7918)
  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: pRRL.EF1a.HA.NLS.Sce(opt).T2A.BFP
FEATURES             Location/Qualifiers
     source          1..7918
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        8..234
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             235..415
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    462..587
                     /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        1988..2199
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2208..2217
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2223..2249
                     /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             2223..2249
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2259..2279
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3000..3053
                     /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             3054..3755
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    3769..4357
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3822..3842)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4240..4251)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4444..4677
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4749..4870
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4786..4805)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4840..4859
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4910..5045
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5065..5084)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5066..5084)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5094..5111)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5094..5110)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5103..5125)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5252..5707
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5339..5358)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5549..5570
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5733..5837
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5838..5906,5907..6698)
                     /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     complement(6056..6075)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6869..7457
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7358..7377
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7611..7628
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7745..7766
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7781..7786,7787..7804,7805..7811)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7819..7835
                     /label=lac operator
                     /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-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7824..7846
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7843..7859
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7843..7859
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7878..7898
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7880..7898
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag
       61 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg
      121 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact
      181 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct
      241 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      301 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      361 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
      421 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
      481 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
      541 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
      601 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
      661 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
      721 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
      781 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
      841 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
      901 aaagtaagac caccgcacag caagcggccg 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 aataaatctc 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 atcacgagac tagcctcgaa attcgagcta gctgcagagg ctccggtgcc
     1981 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
     2041 aattgaaccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
     2101 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
     2161 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg atctgccgcc accatgggcg
     2221 tataccccta cgacgtgccc gactacgccc ccgggccccc taagaaaaag aggaaggtga
     2281 agaacatcaa gaagaaccag gtcatgaacc tgggccccaa cagcaagctg ctgaaggagt
     2341 acaagagcca gctgatcgag ctgaacatcg agcagttcga ggccggcatc ggcctgatcc
     2401 tgggcgacgc ctacatcagg agcagggacg agggcaagac ctactgcatg cagttcgagt
     2461 ggaagaacaa ggcctacatg gaccacgtgt gcctgctgta cgaccagtgg gtgctgagcc
     2521 ccccccacaa gaaggagagg gtgaaccacc tgggcaacct ggtcatcacc tggggcgccc
     2581 agaccttcaa gcaccaggcc ttcaacaagc tggccaacct gttcatcgtg aacaacaaga
     2641 agaccatccc caacaacctg gtggagaact acctgacccc catgagcctg gcctactggt
     2701 tcatggacga cggcggcaag tgggactaca acaagaacag caccaacaag agcatcgtgc
     2761 tgaacaccca gagcttcacc ttcgaggagg tggagtacct ggtgaagggc ctgaggaaca
     2821 agttccagct gaactgctac gtgaagatca acaagaacaa gcccatcatc tacatcgaca
     2881 gcatgagcta cctgatcttc tacaacctga tcaagcccta cctgatcccc cagatgatgt
     2941 acaagctgcc caacaccatc agcagcgaga ccttcctgaa gggcggcggc ggatccggtg
     3001 agggcagagg aagtcttcta acatgcggtg acgtggagga gaatccgggc cccatgagcg
     3061 agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc
     3121 acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa
     3181 tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc
     3241 tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt
     3301 ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga
     3361 ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag
     3421 gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct
     3481 tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga
     3541 agctcgtggg cgggagccat ctgatcgcaa acatcaagac cacatataga tccaagaaac
     3601 ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca
     3661 aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg
     3721 acctccctag caaactgggg cacaagctta attgattcta gagtcgacaa tcaacctctg
     3781 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     3841 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     3901 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     3961 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     4021 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     4081 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     4141 aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc
     4201 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     4261 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     4321 cagacgagtc ggatctccct ttgggccgcc tccccgcctg gaattcgagc tcggtacctt
     4381 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     4441 gactggaagg gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt
     4501 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
     4561 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
     4621 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagta
     4681 gtagttcatg tcatcttatt attcagtatt tataacttgc aaagaaatga atatcagaga
     4741 gtgagaggaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4801 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4861 atgtatctta tcatgtctgg ctctagctat cccgccccta actccgccca tcccgcccct
     4921 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4981 agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg
     5041 aggcctaggg acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc
     5101 gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca
     5161 gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc
     5221 caacagttgc gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg
     5281 gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct
     5341 cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta
     5401 aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa
     5461 cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct
     5521 ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc
     5581 aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg
     5641 ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt
     5701 acaatttagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     5761 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     5821 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     5881 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     5941 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     6001 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     6061 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     6121 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     6181 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     6241 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     6301 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     6361 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     6421 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     6481 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     6541 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     6601 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     6661 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     6721 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     6781 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     6841 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     6901 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     6961 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     7021 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     7081 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     7141 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     7201 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     7261 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     7321 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     7381 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     7441 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     7501 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     7561 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     7621 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     7681 ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg
     7741 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg
     7801 ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc
     7861 atgattacgc caagcgcgca attaaccctc actaaaggga acaaaagctg gagctgca
//
