LOCUS       sequence-372907-        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 (Sce target) E...
FEATURES             Location/Qualifiers
     source          1..7887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     177..196
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     430..447
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    520..744
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     733..750
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     protein_bind    955..988
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1064..1652
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             1170..1181
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     1579..1599
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(1666..2265)
                     /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(1749..1769)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     2246..2265
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(2281..2492)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             complement(2519..3226)
                     /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(2797..2816)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3066..3084
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(3227..3280)
                     /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"
     primer_bind     complement(3337..3358)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3686..3705
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3966..3987
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      4029..4038
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(4053..4459)
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     misc_feature    4488..4605
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     CDS             complement(4872..4916)
                     /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    5101..5334
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    5826..5951
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             5998..6178
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(6179..6405)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     promoter        6439..6543
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6544..6612,6613..7404)
                     /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(6762..6781)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(7575..7887,1..276)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
       61 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
      121 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
      181 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
      241 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
      301 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
      361 tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg
      421 accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct
      481 ctccccgcgc gttggccgat tcattaatgc agctgcaggc catagagccc accgcatccc
      541 cagcatgcct gctattgtct tcccaatcct cccccttgct gtcctgcccc accccacccc
      601 ccagaataga atgacaccta ctcagacaat gcgatgcaat ttcctcattt tattaggaaa
      661 ggacagtggg agtggcacct tccagggtca aggaaggcac gggggagggg caaacaacag
      721 atggctggca actagaaggc acagtcgagg ctggtcgagg caagctttat tgaggcttaa
      781 gcagtgggtt ccctagttag ccagagagct cccaggctca gatctggtct aaccagagag
      841 acccagtaca agcaaaaagc aatggttaca aataaagcaa tagcatcaca aatttcacaa
      901 ataaacatat ggttacaaat aaagcaatag catcacaaat ttcacaaata aattataact
      961 tcgtataatg tatgctatac gaagttatga tatcttgtct tcgttgggag tgaattagcc
     1021 cttccagtcc ccccttttct tttaaaaagg taccgaattc caggcgggga ggcggcccaa
     1081 agggagatcc gactcgtctg agggcgaagg cgaagacgcg gaagaggccg cagagccggc
     1141 agcaggccgc gggaaggaag gtccgctgga ttgagggccg aagggacgta gcagaaggac
     1201 gtcccgcgca gaatccaggt ggcaacacag gcgagcagcc atggaaagga cgtcagcttc
     1261 cccgacaaca ccacggaatt gtcagtgccc aacagccgag cccctgtcca gcagcgggca
     1321 aggcaggcgg cgatgagttc cgccgtggca atagggaggg ggaaagcgaa agtcccggaa
     1381 aggagctgac aggtggtggc aatgccccaa ccagtggggg ttgcgtcagc aaacacagtg
     1441 cacaccacgc cacgttgcct gacaacgggc cacaactcct cataaagaga cagcaaccag
     1501 gatttataca aggaggagaa aatgaaagcc atacgggaag caatagcatg atacaaaggc
     1561 attaaagcag cgtatccaca tagcgtaaaa ggagcaacat agttaagaat accagtcaat
     1621 ctttcacaaa ttttgtaatc cagaggttga ttgtcgacac tagattcagg caccgggctt
     1681 gcgggtcatg caccaggtgc gcggtccttc gggcacctcg acgtcggcgg tgacggtgaa
     1741 gccgagccgc tcgtagaagg ggaggttgcg gggcgcggag gtctccagga aggcgggcac
     1801 cccggcgcgc tcggccgcct ccactccggg gagcacgacg gcgctgccca gacccttgcc
     1861 ctggtggtcg ggcgagacgc cgacggtggc caggaaccac gcgggctcct tgggccggtg
     1921 cggcgccagg aggccttcca tctgttgctg cgcggccagc cgggaaccgc tcaactcggc
     1981 catgcgcggg ccgatctcgg cgaacaccgc ccccgcttcg acgctctccg gcgtggtcca
     2041 gaccgccacc gcggcgccgt cgtccgcgac ccacaccttg ccgatgtcga gcccgacgcg
     2101 cgtgaggaag agttcttgca gctcggtgac ccgctcgatg tggcggtccg ggtcgacggt
     2161 gtggcgcgtg gcggggtagt cggcgaacgc ggcggcgagg gtgcgtacgg cccgggggac
     2221 gtcgtcgcgg gtggcgaggc gcaccgtggg cttgtactcg gtcatggtgg ctcgaggatc
     2281 ctgtgttctg gcggcaaacc cgttgcgaaa aagaacgttc acggcgacta ctgcacttat
     2341 atacggttct cccccaccct cgggaaaaag gcggagccag tacacgacat cactttccca
     2401 gtttaccccg cgccaccttc tctaggcacc ggttcaattg ccgacccctc cccccaactt
     2461 ctcggggact gtgggcgatg tgcgctctgc ccactgacgg gcaccggagc ctctgcattc
     2521 acttgtacag ctcgtccatg ccgccggtgg agtggcggcc ctcggcgcgt tcgtactgtt
     2581 ccacgatggt gtagtcctcg ttgtgggagg tgatgtccaa cttgatgttg acgttgtagg
     2641 cgccgggcag ctgcacgggc ttcttggcct tgtaggtggt cttgacctca gcgtcgtagt
     2701 ggccgccgtc cttcagcttc agcctctgct tgatctcgcc cttcagggcg ccgtcctcgg
     2761 ggtacatccg ctcggaggag gcctcccagc ccatggtctt cttctgcatt acggggccgt
     2821 cggaggggaa gttggtgccg cgcagcttca ccttgtagat gaactcgccg tcctgcagag
     2881 aggagtcctg ggtcacggtc accacgccgc cgtcctcgaa gttcatcacg cgctcccact
     2941 tgaagccctc ggggaaggac agcttcaagt agtcggggat gtcggcgggg tgcttcacgt
     3001 aggccttgga gccgtacatg aactgagggg acaggatgtc ccaggcgaag ggcagggggc
     3061 cacccttggt caccttcagc ttggcggtct gggtgccctc gtaggggcgg ccctcgccct
     3121 cgccctcgat ctcgaactcg tggccgttca cggagccctc catgtgcacc ttgaagcgca
     3181 ggaactcctt gatgatggcg gagttatcct cctcgccctt gctcacaggg cccggattct
     3241 cctccacgtc accgcatgtt agaagacttc ctctgccctc accggatcca gttacttgta
     3301 cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg
     3361 atcgcgcttc tcgttggggt ctttgctcag ggcggactgg gtgctcaggt agtggttgtc
     3421 gggcagcagc acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg
     3481 cacgctgccg tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg
     3541 cttgtcggcc atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag
     3601 gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc
     3661 gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt
     3721 gcgctcctgg acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg
     3781 gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca
     3841 gggcacgggc agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtgag
     3901 ctcttatttg cgtagggata acagggtaat gtcgaggccg gacacgctga acttgtggcc
     3961 gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc
     4021 cttgctcacc atggtggcgc tcgaggatcg atccgggcga ctcagtctgt cggaggactg
     4081 gcgcgccgag tgaggggttg tgagctcttt tatagagctc gggaagcaga agcgcgcgaa
     4141 cagaagcgag aagcaggctg attggttaat tcaaataagg cgcagggtca tttcaggtcc
     4201 ttgggggagc ctggaaacat ctgatgggtc ttaagaaact gctgagggtt gggccatatc
     4261 tggggaccat ctgttcttgg cctcgggccg gggccgaaac tgcggtgacc atctgttctt
     4321 ggccccgggc cggggccgaa actgctcacc gcagatatcc tgtttggccc aacgttagct
     4381 attttcatgt acccgccctt gatctgaact tctctattct tggtttggta tttttccatg
     4441 ccttgcaaaa tggcgttact gcagctagca cgtgacgcgt aatcgataaa attttgaatt
     4501 tttgtaattt gtttttgtaa ttctttagtt tgtatgtctg ttgctattat gtctactatt
     4561 ctttcccctg cactgtaccc cccaatcccc ccttttcttt taaaagttaa ccgataccgt
     4621 cgagatccgt tcactaatcg aatggatctg tctctgtctc tctctccacc ttcttcttct
     4681 attccttcgg gcctgtcggg tcccctcggg gttgggaggt gggtctgaaa cgataatggt
     4741 gaatatccct gcctaactct attcactata gaaagtacag caaaaactat tcttaaacct
     4801 accaagcctc ctactatcat tatgaataat tttatatacc acagccaatt tgttatgtta
     4861 aaccaattcc acaaacttgc ccatttatct aattccaata attcttgttc attcttttct
     4921 tgctggtttt gcgattcttc aattaaggag tgtattaagc ttgtgtaatt gttaatttct
     4981 ctgtcccact ccatccaggt cgtgtgattc caaatctgtt ccagagattc attactccaa
     5041 ctagcattcc aaggcacagc agtggtgcaa atgagttttc cagagcaacc ccaaatcccc
     5101 aggagctgtt gatcctttag gtatctttcc acagccagga ttcttgcctg gagctgcttg
     5161 atgccccaga ctgtgagttg caacagatgc tgttgcgcct caatagccct cagcaaattg
     5221 ttctgctgct gcactatacc agacaataat tgtctggcct gtaccgtcag cgtcattgac
     5281 gctgcgccca tagtgcttcc tgctgctccc aagaacccaa ggaacaaagc tcctattccc
     5341 actgctcttt tttctctctg caccactctt ctctttgcct tggtgggtgc tactcctaat
     5401 ggttcaattt ttactacttt atatttatat aattcacttc tccaattgtc cctcatatct
     5461 cctcctccag gtctgaagat cagggccgct tgctgtgcgg tggtcttact tttgttttgc
     5521 tcttcctcta tcttgtctaa agcttccttg gtgtctttta tctctatcct ttgatgcaca
     5581 caatagaggg ttgctactgt attatataat gatctaagtt cttctgatcc tgtctgaagg
     5641 gatggttgta gctgtcccag tatttgtcta cagccttctg atgtttctaa caggccagga
     5701 ttaactgcga atcgttctag ctccctgctt gcccatacta tatgttttaa tttatatttt
     5761 ttctttcccc ctggccttaa ccgaattttt tcccatcgcg atctaattct cccccgctta
     5821 atactgacgc tctcgcaccc atctctctcc ttctagcctc cgctagtcaa aatttttggc
     5881 gtactcacca gtcgccgccc ctcgcctctt gccgtgcgcg cttcagcaag ccgagtcctg
     5941 cgtcgagaga gctcctctgg tttccctttc gctttcaagt ccctgttcgg gcgccactgc
     6001 tagagatttt ccacactgac taaaagggtc tgagggatct ctagttacca gagtcacaca
     6061 acagacgggc acacactact tgaagcactc aaggcaagct ttattgaggc ttaagcagtg
     6121 ggttccctag ttagccagag agctcccagg ctcagatctg gtctaaccag agagacccgt
     6181 ttatgtatcg agctaggcac ttaaatacaa tatctctgca atgcggcaat tcagtggttc
     6241 gtccaatcca tgtcagaccc gtctgttgcc ttcctaataa ggcacgatcg taccacctta
     6301 cttccaccaa tcggcatgca cggtgctttt tctctccttg taaggcatgt tgctaactca
     6361 tcgttaccat gttgcaagac tacaagagta ttgcataaga ctacattgac gtcaggtggc
     6421 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     6481 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     6541 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     6601 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     6661 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     6721 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     6781 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     6841 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     6901 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     6961 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     7021 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     7081 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     7141 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     7201 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     7261 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     7321 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     7381 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     7441 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     7501 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     7561 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     7621 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     7681 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     7741 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     7801 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     7861 tagttaccgg ataaggcgca gcggtcg
//
