LOCUS       sequence-14415-e        7887 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 7887)
  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 7887)
  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 Traffic Light Reporter 1.1 (Ani target) E...
FEATURES             Location/Qualifiers
     source          1..7887
                     /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"
     primer_bind     complement(2427..2448)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2709..2728)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3056..3077
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3134..3187
                     /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             3188..3895
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNSAIIKEFLRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(3330..3348)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3598..3617
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        3922..4133
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             4149..4748
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4149..4168)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4645..4665
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     misc_feature    4762..5350
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4815..4835)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5233..5244)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(5426..5459)
                     /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(5664..5681)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5670..5894
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5967..5984)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6138..6726)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6218..6237)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6897..7688,7689..7757))
                     /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     7520..7539
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7758..7862)
                     /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 ggctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     2461 agttcagcgt gtccggcctc gaccgtgagg aggtttctct gtaaagctaa gagctcacct
     2521 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     2581 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     2641 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     2701 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     2761 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     2821 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     2881 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     2941 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     3001 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     3061 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
     3121 aactggatcc ggtgagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc
     3181 gggccctgtg agcaagggcg aggaggataa ctccgccatc atcaaggagt tcctgcgctt
     3241 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
     3301 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
     3361 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
     3421 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
     3481 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctctctgca
     3541 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
     3601 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
     3661 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
     3721 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
     3781 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
     3841 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agtgaatgca
     3901 gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
     3961 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
     4021 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
     4081 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggatcctc
     4141 gagccaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtccccc
     4201 gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg
     4261 acccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg
     4321 ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca
     4381 cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt
     4441 tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc
     4501 ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg
     4561 gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg
     4621 ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg
     4681 tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg
     4741 gtgcctgaat ctagtgtcga caatcaacct ctggattaca aaatttgtga aagattgact
     4801 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     4861 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     4921 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     4981 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     5041 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     5101 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaagctg
     5161 acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     5221 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     5281 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     5341 gcctccccgc ctggaattcg gtaccttttt aaaagaaaag gggggactgg aagggctaat
     5401 tcactcccaa cgaagacaag atatcataac ttcgtatagc atacattata cgaagttata
     5461 atttatttgt gaaatttgtg atgctattgc tttatttgta accatatgtt tatttgtgaa
     5521 atttgtgatg ctattgcttt atttgtaacc attgcttttt gcttgtactg ggtctctctg
     5581 gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc
     5641 tcaataaagc ttgcctcgac cagcctcgac tgtgccttct agttgccagc catctgttgt
     5701 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     5761 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     5821 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     5881 ggtgggctct atggcctgca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5941 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     6001 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     6061 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     6121 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     6181 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     6241 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     6301 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6361 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6421 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6481 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6541 ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct
     6601 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6661 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6721 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6781 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6841 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6901 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6961 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     7021 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     7081 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     7141 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     7201 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     7261 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     7321 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     7381 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     7441 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7501 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7561 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7621 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     7681 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7741 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7801 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     7861 cgcacatttc cccgaaaagt gccacct
//
