LOCUS       sequence-14419-e        8200 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 8200)
  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 8200)
  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 sEF1a HA.NLS.Sce(opt).T2A.IFP
FEATURES             Location/Qualifiers
     source          1..8200
                     /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        1986..2197
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     regulatory      2206..2215
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2221..2247
                     /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             2221..2247
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2257..2277
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2998..3051
                     /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             3052..4017
                     /codon_start=1
                     /product="bacteriophytochrome-based infrared fluorescent 
                     protein (Shu et al., 2009)"
                     /label=IFP1.1
                     /translation="MARDPLPFFPPLYLGGPEITTENCEREPIHIPGSIQPHGALLTAD
                     GHSGEVLQVSLNAATFLGQEPTVLRGQTLAALLPEQWPALQAALPPGCPDALQYRATLD
                     WPAAGHLSLTVHRVAELLILEFEPTEAWDSIGPHALRNAMFALESAPNLRALAEVATQT
                     VRELTGFDRVMLYKFAPDATGEMIAEARREGMQAFLGHRFPASHTPAQARALYTRHLLR
                     LTADTRAAAVPLDPVLNPQTNAPTPLGGAVLRATSPMHMQYLRNMGVGSSLSVSVVVGG
                     QLWGLIVCHHQTPYVLPPDLRTTLEELGRKLSGQVQRKEA"
     misc_feature    4045..4633
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4098..4118)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4516..4527)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4720..4953
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5025..5146
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5062..5081)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5116..5135
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      5186..5321
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(5341..5360)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5342..5360)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5370..5387)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5370..5386)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5379..5401)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5531..5986
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5618..5637)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5828..5849
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6015..6119
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6120..6188,6189..6980)
                     /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(6338..6357)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7151..7739
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7640..7659
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7893..7910
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8027..8048
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8063..8068,8069..8086,8087..8093)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8101..8117
                     /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     8106..8128
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8125..8141
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8125..8141
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     8160..8180
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        8162..8180
                     /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 tgcagaggct ccggtgcccg
     1981 tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag gggtcggcaa
     2041 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg
     2101 gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa
     2161 cgttcttttt cgcaacgggt ttgccgccag aacacaggat ctgccgccac catgggcgta
     2221 tacccctacg acgtgcccga ctacgccccc gggcccccta agaaaaagag gaaggtgaag
     2281 aacatcaaga agaaccaggt catgaacctg ggccccaaca gcaagctgct gaaggagtac
     2341 aagagccagc tgatcgagct gaacatcgag cagttcgagg ccggcatcgg cctgatcctg
     2401 ggcgacgcct acatcaggag cagggacgag ggcaagacct actgcatgca gttcgagtgg
     2461 aagaacaagg cctacatgga ccacgtgtgc ctgctgtacg accagtgggt gctgagcccc
     2521 ccccacaaga aggagagggt gaaccacctg ggcaacctgg tcatcacctg gggcgcccag
     2581 accttcaagc accaggcctt caacaagctg gccaacctgt tcatcgtgaa caacaagaag
     2641 accatcccca acaacctggt ggagaactac ctgaccccca tgagcctggc ctactggttc
     2701 atggacgacg gcggcaagtg ggactacaac aagaacagca ccaacaagag catcgtgctg
     2761 aacacccaga gcttcacctt cgaggaggtg gagtacctgg tgaagggcct gaggaacaag
     2821 ttccagctga actgctacgt gaagatcaac aagaacaagc ccatcatcta catcgacagc
     2881 atgagctacc tgatcttcta caacctgatc aagccctacc tgatccccca gatgatgtac
     2941 aagctgccca acaccatcag cagcgagacc ttcctgaagg gcggcggcgg atccggtgag
     3001 ggcagaggaa gtcttctaac atgcggtgac gtggaggaga atccgggccc catggctcgg
     3061 gaccctctgc cattctttcc acctctgtac ctgggcggcc ctgagattac aaccgagaac
     3121 tgcgagagag agcctatcca cattcctggg tccatccagc cacacggggc tctgctcaca
     3181 gctgacggcc actccggaga ggtgctccaa gtgtccctga atgccgctac cttcctgggc
     3241 caggagccta ctgtgctgcg ggggcagacc ctggctgccc tgctccccga gcagtggcca
     3301 gccctgcagg cagccctgcc cccaggatgt ccagatgccc tccaatacag ggccaccctc
     3361 gactggccag ctgctgggca cctcagcctg actgtgcatc gggtggctga actcctgatc
     3421 ctggagttcg aacctaccga ggcctgggac agcattggcc ctcacgccct gaggaacgcc
     3481 atgtttgctc tggaaagcgc tccaaacctg cgggctctgg ccgaagtcgc aacacaaact
     3541 gtgagagaac tgactggctt cgatcgggtg atgctgtaca aatttgcccc tgacgccact
     3601 ggagagatga ttgctgaggc cagacgggag ggcatgcagg cttttctggg ccacaggttt
     3661 cccgcatccc acacccctgc acaagctaga gccctctaca caagacacct gctccggctg
     3721 accgcagaca ccagggctgc agcagtgccc ctcgaccccg tgctgaatcc ccagacaaat
     3781 gctcctacac ctctgggcgg agctgtcctc agagctacat ccccaatgca catgcagtac
     3841 ctgaggaata tgggagtggg ctcctccctg agcgtcagcg tcgtggtcgg cggccagctg
     3901 tggggactga ttgtctgcca ccatcagaca ccctacgtgc tgccaccaga tctgcggacc
     3961 accctggagg agctggggag aaagctgtcc gggcaggtgc agaggaaaga agcctgatct
     4021 agaattggga catctagtgt cgacaatcaa cctctggatt acaaaatttg tgaaagattg
     4081 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     4141 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     4201 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     4261 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     4321 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     4381 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaag
     4441 ctgacgtcct ttccatggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     4501 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     4561 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     4621 gccgcctccc cgcctggaat tcgagctcgg tacctttaag accaatgact tacaaggcag
     4681 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     4741 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     4801 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     4861 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     4921 gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc
     4981 agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag
     5041 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5101 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct
     5161 agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     5221 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     5281 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg
     5341 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     5401 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     5461 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     5521 gaatggcgcg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     5581 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     5641 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5701 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     5761 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5821 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     5881 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5941 caaaaattta acgcgaattt taacaaaata ttaacgttta caatttccca ggtggcactt
     6001 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     6061 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     6121 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     6181 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     6241 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     6301 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     6361 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     6421 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     6481 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     6541 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     6601 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     6661 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     6721 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     6781 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc
     6841 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     6901 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     6961 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     7021 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     7081 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     7141 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     7201 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     7261 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     7321 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     7381 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     7441 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     7501 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     7561 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     7621 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     7681 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     7741 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     7801 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     7861 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     7921 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
     7981 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
     8041 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
     8101 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg
     8161 caattaaccc tcactaaagg gaacaaaagc tggagctgca
//
