LOCUS       sequence-174194-        3504 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  visualization and quantifying the out-of-frame mutations from genome
            editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3504)
  AUTHORS   Fu L, Wen L, Luu N, Shi YB
  TITLE     A simple and efficient method to visualize and quantify the 
            efficiency of chromosomal mutations from genome editing.
  JOURNAL   Sci Rep. 2016 Oct 17;6:35488. doi: 10.1038/srep35488.
  PUBMED    27748423
REFERENCE   2  (bases 1 to 3504)
  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"; journalName: "Sci Rep."; 
            date: "2016-10-17"; volume: "6:35488. doi"; pages: " 
            10.1038/srep35488"
COMMENT     Sequence Label: pLacZa-GFP
FEATURES             Location/Qualifiers
     source          1..3504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(190..1287)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDAASSTLEDPRVPSSNS
                     GDAWGSLCRDLCGNSTGRRFTGTMRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGD
                     ATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQE
                     RTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITA
                     DKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNE
                     KRDHMVLLEFVTAAGITHGMDELYK"
     CDS             complement(193..906)
                     /codon_start=1
                     /product="GFP variant that folds robustly even when fused 
                     to poorly folded proteins (Pedelacq et al., 2006)"
                     /label=superfolder GFP
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKA
                     NFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     914..930
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1283..1299)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1283..1299)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1296..1318)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1307..1323
                     /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)."
     promoter        complement(join(1331..1337,1338..1355,1356..1361))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1376..1397
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1514..1531)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1685..2273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1765..1784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2444..3235,3236..3304))
                     /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     3067..3086
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3305..3409)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3477..3495
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatcat catttgtaca gttcatccat accatgcgtg atgcccgctg cggttacgaa
      241 ctccagcaga accatatgat cgcgtttctc gttcggatct ttagacagaa cgctttgcgt
      301 gctcagatag tgattgtctg gcagcagaac aggaccatca ccgattggag tgttttgctg
      361 gtagtgatca gccagctgca cgctgccatc ctccacgttg tggcgaattt taaaattcgc
      421 tttaatgcca tttttttgtt tatcggcggt gatgtaaaca ttgtggctgt taaaattgta
      481 ttccagctta tggcccagga tattgccgtc ttctttaaag tcaatgcctt tcagctcaat
      541 gcggtttacc agggtatcgc cttcaaattt cacttccgca cgcgttttgt acgtgccgtc
      601 atccttaaag gaaatcgtgc gttcctgcac atagccttcc ggcatggcgg acttgaagaa
      661 gtcatgctgc ttcatatggt ccggataacg agcaaagcac tgaacaccat aagtcagcgt
      721 cgttaccaga gtcggccaag gtaccggcag tttaccagta gtacagatga acttcagcgt
      781 cagtttacca ttagttgcgt caccttcacc ctcgccacgc acggaaaact tatgaccgtt
      841 gacatcacca tccagttcca ccagaatagg gacgacacca gtgaacagct cctcgccttt
      901 acgcatggta ccggtaaaac gacggccagt tgaatttcca cacagatccc ggcacaaact
      961 tccccatgca tctcccgaat tcgagctcgg tacccgggga tcctctagag tcgacgatgc
     1021 ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac cgcacagatg
     1081 cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca actgttggga
     1141 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc
     1201 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta acactaccgc
     1261 tagcaagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     1321 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     1381 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     1441 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     1501 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     1561 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     1621 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1681 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1741 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1801 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     1861 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     1921 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     1981 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2041 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     2101 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     2161 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     2221 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     2281 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     2341 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     2401 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     2461 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     2521 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     2581 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     2641 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     2701 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     2761 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     2821 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     2881 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     2941 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     3001 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     3061 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     3121 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     3181 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     3241 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     3301 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     3361 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     3421 gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata
     3481 ggcgtatcac gaggcccttt cgtc
//
