LOCUS       sequence-14420-e        7985 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 7985)
  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 7985)
  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 SFFV d20GFP.T2A.mTagBFP Donor
FEATURES             Location/Qualifiers
     source          1..7985
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             236..416
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    460..585
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1078..1311
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1496..1540
                     /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    1807..1924
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1965..2371
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      2386..2395
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2392..2394,2395..2397,2398..2910)
                     /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(2437..2458)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2698..2717)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             3061..3114
                     /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             3115..3816
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    3830..4418
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3883..3903)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4301..4312)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4505..4738
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4810..4931
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4847..4866)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4901..4920
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4971..5106
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5126..5145)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5127..5145)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5155..5172)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5155..5171)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5164..5186)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5316..5771
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5403..5422)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5613..5634
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5800..5904
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5905..5973,5974..6765)
                     /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(6123..6142)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6936..7524
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7425..7444
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7678..7695
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7812..7833
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7848..7853,7854..7871,7872..7878)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7886..7902
                     /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     7891..7913
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7910..7926
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7910..7926
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7945..7965
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7947..7965
                     /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 ctcgatacaa taaacgggtc
      241 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      301 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      361 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
      421 cgcccgaaca gggacctgaa agcgaaaggg aaaccagagc tctctcgacg caggactcgg
      481 cttgctgaag cgcgcacggc aagaggcgag gggcggcgac tggtgagtac gccaaaaatt
      541 ttgactagcg gaggctagaa ggagagagat gggtgcgaga gcgtcagtat taagcggggg
      601 agaattagat cgcgatggga aaaaattcgg ttaaggccag ggggaaagaa aaaatataaa
      661 ttaaaacata tagtatgggc aagcagggag ctagaacgat tcgcagttaa tcctggcctg
      721 ttagaaacat cagaaggctg tagacaaata ctgggacagc tacaaccatc ccttcagaca
      781 ggatcagaag aacttagatc attatataat acagtagcaa ccctctattg tgtgcatcaa
      841 aggatagaga taaaagacac caaggaagct ttagacaaga tagaggaaga gcaaaacaaa
      901 agtaagacca ccgcacagca agcggccgct gatcttcaga cctggaggag gagatatgag
      961 ggacaattgg agaagtgaat tatataaata taaagtagta aaaattgaac cattaggagt
     1021 agcacccacc aaggcaaaga gaagagtggt gcagagagaa aaaagagcag tgggaatagg
     1081 agctttgttc cttgggttct tgggagcagc aggaagcact atgggcgcag cctcaatgac
     1141 gctgacggta caggccagac aattattgtc tggtatagtg cagcagcaga acaatttgct
     1201 gagggctatt gaggcgcaac agcatctgtt gcaactcaca gtctggggca tcaagcagct
     1261 ccaggcaaga atcctggctg tggaaagata cctaaaggat caacagctcc tggggatttg
     1321 gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta gttggagtaa
     1381 taaatctctg gaacagattt ggaatcacac gacctggatg gagtgggaca gagaaattaa
     1441 caattacaca agcttaatac actccttaat tgaagaatcg caaaaccagc aagaaaagaa
     1501 tgaacaagaa ttattggaat tagataaatg ggcaagtttg tggaattggt ttaacataac
     1561 aaattggctg tggtatataa aattattcat aatgatagta ggaggcttgg taggtttaag
     1621 aatagttttt gctgtacttt ctatagtgaa tagagttagg cagggatatt caccattatc
     1681 gtttcagacc cacctcccaa ccccgagggg acccgacagg cccgaaggaa tagaagaaga
     1741 aggtggagag agagacagag acagatccat tcgattagtg aacggatctc gacggtatcg
     1801 gttaactttt aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga
     1861 cataatagca acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa
     1921 ttttatcgat cacgagacta gcctcgaaat tcgagctagc tgcagtaacg ccattttgca
     1981 aggcatggaa aaataccaaa ccaagaatag agaagttcag atcaagggcg ggtacatgaa
     2041 aatagctaac gttgggccaa acaggatatc tgcggtgagc agtttcggcc ccggcccggg
     2101 gccaagaaca gatggtcacc gcagtttcgg ccccggcccg aggccaagaa cagatggtcc
     2161 ccagatatgg cccaaccctc agcagtttct taagacccat cagatgtttc caggctcccc
     2221 caaggacctg aaatgaccct gcgccttatt tgaattaacc aatcagcctg cttctcgctt
     2281 ctgttcgcgc gcttctgctt cccgagctct ataaaagagc tcacaacccc tcactcggcg
     2341 cgccagtcct ccgacagact gagtcgcccg gatcgatcct cgagcgccac catggtgagc
     2401 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2461 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     2521 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     2581 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     2641 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2701 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2761 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     2821 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     2881 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     2941 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3001 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cggatccggt
     3061 gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatccggg ccccatgagc
     3121 gagctgatta aggagaacat gcacatgaag ctgtacatgg agggcaccgt ggacaaccat
     3181 cacttcaagt gcacatccga gggcgaaggc aagccctacg agggcaccca gaccatgaga
     3241 atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg acatcctggc tactagcttc
     3301 ctctacggca gcaagacctt catcaaccac acccagggca tccccgactt cttcaagcag
     3361 tccttccctg agggcttcac atgggagaga gtcaccacat acgaagacgg gggcgtgctg
     3421 accgctaccc aggacaccag cctccaggac ggctgcctca tctacaacgt caagatcaga
     3481 ggggtgaact tcacatccaa cggccctgtg atgcagaaga aaacactcgg ctgggaggcc
     3541 ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag gcagaaacga catggccctg
     3601 aagctcgtgg gcgggagcca tctgatcgca aacatcaaga ccacatatag atccaagaaa
     3661 cccgctaaga acctcaagat gcctggcgtc tactatgtgg actacagact ggaaagaatc
     3721 aaggaggcca acaacgagac ctacgtcgag cagcacgagg tggcagtggc cagatactgc
     3781 gacctcccta gcaaactggg gcacaagctt aattgattct agagtcgaca atcaacctct
     3841 ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct
     3901 atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta tggctttcat
     3961 tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt ggcccgttgt
     4021 caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg gttggggcat
     4081 tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta ttgccacggc
     4141 ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt tgggcactga
     4201 caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg cctgtgttgc
     4261 cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca atccagcgga
     4321 ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc gccttcgccc
     4381 tcagacgagt cggatctccc tttgggccgc ctccccgcct ggaattcgag ctcggtacct
     4441 ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa agaaaagggg
     4501 ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc ttgtactggg
     4561 tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg
     4621 cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt
     4681 gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt
     4741 agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg aatatcagag
     4801 agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4861 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4921 aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc atcccgcccc
     4981 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     5041 cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg
     5101 gaggcctagg gacgtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc
     5161 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     5221 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     5281 ccaacagttg cgcagcctga atggcgaatg gcgcgacgcg ccctgtagcg gcgcattaag
     5341 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     5401 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     5461 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     5521 aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
     5581 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
     5641 actcaaccct atctcggtct attcttttga tttataaggg attttgccga tttcggccta
     5701 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
     5761 gtttacaatt tcccaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     5821 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     5881 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     5941 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     6001 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     6061 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     6121 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     6181 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     6241 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     6301 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     6361 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     6421 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     6481 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     6541 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     6601 tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag
     6661 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     6721 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     6781 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     6841 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     6901 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     6961 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     7021 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     7081 gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     7141 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     7201 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     7261 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     7321 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     7381 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     7441 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     7501 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     7561 ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac
     7621 cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc
     7681 gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt
     7741 tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag
     7801 cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg
     7861 cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc
     7921 tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca aaagctggag
     7981 ctgca
//
