LOCUS       sequence-14416-e        7995 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 7995)
  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 7995)
  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 (Sce target) E...
FEATURES             Location/Qualifiers
     source          1..7995
                     /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             4152..4853
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     misc_feature    4870..5458
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4923..4943)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5341..5352)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     protein_bind    complement(5534..5567)
                     /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(5772..5789)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5778..6002
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6075..6092)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6246..6834)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6326..6345)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7005..7796,7797..7865))
                     /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     7628..7647
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7866..7970)
                     /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 gacattaccc tgttatccct acgcaaataa 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 caggatccac
     4141 cggtcgccac 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 tagtgaatct
     4861 agagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     4921 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
     4981 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
     5041 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
     5101 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
     5161 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
     5221 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca
     5281 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
     5341 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
     5401 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct
     5461 ggaattcggt acctttttaa aagaaaaggg gggactggaa gggctaattc actcccaacg
     5521 aagacaagat atcataactt cgtatagcat acattatacg aagttataat ttatttgtga
     5581 aatttgtgat gctattgctt tatttgtaac catatgttta tttgtgaaat ttgtgatgct
     5641 attgctttat ttgtaaccat tgctttttgc ttgtactggg tctctctggt tagaccagat
     5701 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     5761 gcctcgacca gcctcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc
     5821 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga
     5881 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga
     5941 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat
     6001 ggcctgcagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     6061 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     6121 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     6181 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     6241 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     6301 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     6361 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6421 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6481 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6541 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6601 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6661 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     6721 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6781 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6841 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6901 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6961 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     7021 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     7081 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     7141 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     7201 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     7261 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     7321 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     7381 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7441 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7501 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7561 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7621 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7681 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7741 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7801 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7861 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7921 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7981 cgaaaagtgc cacct
//
