LOCUS       sequence-160345-        5648 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express copGFP reporter for HDR-induced knock-in.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5648)
  AUTHORS   He X, Tan C, Wang F, Wang Y, Zhou R, Cui D, You W, Zhao H, Ren J, 
            Feng B
  TITLE     Knock-in of large reporter genes in human cells via 
            CRISPR/Cas9-induced homology-dependent and independent DNA repair.
  JOURNAL   Nucleic Acids Res. 2016 May 19;44(9):e85. doi: 10.1093/nar/gkw064. 
            Epub 2016 Feb 4.
  PUBMED    26850641
REFERENCE   2  (bases 1 to 5648)
  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: "Nucleic 
            Acids Res."; date: "2016-05-19"; volume: "44(9):e85. doi"; pages: " 
            10.1093/nar/gkw064. Epub 2016 Feb 4"
COMMENT     Sequence Label: 2a-copGFP(+HA)
FEATURES             Location/Qualifiers
     source          1..5648
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     677..693
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     677..693
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             1618..1674
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             1699..2361
                     /codon_start=1
                     /product="green fluorescent protein 2 from Pontellina 
                     plumata, also known as ppluGFP2 (Shagin et al., 2004)"
                     /label=CopGFP
                     /translation="PAMEIECRITGTLNGVEFELVGGGEGTPKQGRMTNKMKSTKGALT
                     FSPYLLSHVMGYGFYHFGTYPSGYENPFLHAINNGGYTNTRIEKYEDGGVLHVSFSYRY
                     EAGRVIGDFKVVGTGFPEDSVIFTDKIIRSNATVEHLHPMGDNVLVGSFARTFSLRDGG
                     YYSFVVDSHMHFKSAIHPSILQNGGPMFAFRRVEELHSNTELGIVEYQHAFKTPIAFA"
     primer_bind     complement(3460..3480)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3460..3478)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3499..3515)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3499..3515)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3512..3534)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3523..3539
                     /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(3547..3553,3554..3571,3572..3577))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3592..3613
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3730..3747)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3901..4489)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3981..4000)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4660..5451,5452..5520))
                     /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     5283..5302
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5521..5625)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcca
      661 ccgcggtggc ggccgctcta gaactagtgg atccttacta gccaattgga cacgctcccc
      721 tgacttgcgc cccgctccct ctttctttgc agcaatgcct cctgcaccac caactgctta
      781 gcacccctgg ccaaggtcat ccatgacaac tttggtatcg tggaaggact catggtatga
      841 gagctgggga atgggactga ggctcccacc tttctcatcc aagactggct cctccctgcc
      901 ggggctgcgt gcaaccctgg ggttgggggt tctggggact ggctttccca taatttcctt
      961 tcaaggtggg gagggaggta gaggggtgat gtggggagta cgctgcaggg cctcactcct
     1021 tttgcagacc acagtccatg ccatcactgc cacccagaag actgtggatg gcccctccgg
     1081 gaaactgtgg cgtgatggcc gcggggctct ccagaacatc atccctgcct ctactggcgc
     1141 tgccaaggct gtgggcaagg tcatccctga gctgaacggg aagctcactg gcatggcctt
     1201 ccgtgtcccc actgccaacg tgtcagtggt ggacctgacc tgccgtctag aaaaacctgc
     1261 caaatatgat gacatcaaga aggtggtgaa gcaggcgtcg gagggccccc tcaagggcat
     1321 cctgggctac actgagcacc aggtggtctc ctctgacttc aacagcgaca cccactcctc
     1381 cacctttgac gctggggctg gcattgccct caacgaccac tttgtcaagc tcatttcctg
     1441 gtatgtggct ggggccagag actggctctt aaaaagtgca gggtctggcg ccctctggtg
     1501 gctggctcag aaaaagggcc ctgacaactc ttttcatctt ctaggtatga caacgaattt
     1561 ggctacagca acagggtggt ggacctcatg gcccacatgg cctccaagga gacgcgtgcc
     1621 actaacttct ccctgttgaa acaagcaggg gatgtcgaag agaatcccgg gccaatggag
     1681 agcgacgaga gcggcctgcc cgccatggag atcgagtgcc gcatcaccgg caccctgaac
     1741 ggcgtggagt tcgagctggt gggcggcgga gagggcaccc ccaagcaggg ccgcatgacc
     1801 aacaagatga agagcaccaa aggcgccctg accttcagcc cctacctgct gagccacgtg
     1861 atgggctacg gcttctacca cttcggcacc taccccagcg gctacgagaa ccccttcctg
     1921 cacgccatca acaacggcgg ctacaccaac acccgcatcg agaagtacga ggacggcggc
     1981 gtgctgcacg tgagcttcag ctaccgctac gaggccggcc gcgtgatcgg cgacttcaag
     2041 gtggtgggca ccggcttccc cgaggacagc gtgatcttca ccgacaagat catccgcagc
     2101 aacgccaccg tggagcacct gcaccccatg ggcgataacg tgctggtggg cagcttcgcc
     2161 cgcaccttca gcctgcgcga cggcggctac tacagcttcg tggtggacag ccacatgcac
     2221 ttcaagagcg ccatccaccc cagcatcctg cagaacgggg gccccatgtt cgccttccgc
     2281 cgcgtggagg agctgcacag caacaccgag ctgggcatcg tggagtacca gcacgccttc
     2341 aagaccccca tcgccttcgc cagatcccgc gctcagtcgt ccaattctgc cgtggacggc
     2401 accgccggac ccggctccac cggatctcgc taaagatcaa cccctgccac actcagtccc
     2461 ccaccacact gaatctcccc tcctcacagt tgccatgtag accccttgaa gaggggaggg
     2521 gcctagggag ccgcaccttg tcatgtacca tcaataaagt accctgtgct caaccagtta
     2581 cttgtcctgt cttattctag ggtctggggc agaggggagg gaagctgggc ttgtgtcaag
     2641 gtgagacatt cttgctgggg agggacctgg tatgttctcc tcagactgag ggtagggcct
     2701 ccaaacagcc ttgcttgctt cgagaaccat ttgcttcccg ctcagacgtc ttgagtgcta
     2761 caggaagctg gcaccactac ttcagagaac aaggcctttt cctctcctcg ctccagtcct
     2821 aggctatctg ctgttggcca aacatggaag aagctattct gtgggcggcc ccagggaggc
     2881 tgacaggtgg aggaagtcag ggctcgcact gggctctgac gctgactggt tagtggagct
     2941 cagcctggag ctgagctgca gcgggcaatt ccagcttggc ctccgcagct gtgaggtctt
     3001 gagcacgtgc tctattgctt tctgtgccct cgtgtcttat ctgaggacat cgtggccagc
     3061 ccctaaggtc ttcaagcagg attcatctag gtaaaccaag tacctaaaac catgcccaag
     3121 gcggtaagga ctatataatg tttaaaaatc ggtaaaaatg cccacctcgc atagttttga
     3181 ggaagatgaa ctgagatgtg tcagggtgac ttatttccat catcgtcctt aggggagctt
     3241 gggtaggggc aaggcgtgta gctgggacct aggtccagac ccctggctct gccactgaac
     3301 ggctcagttg ctttgggcag ttactcccgg gcctcacttt gcacgtgtgc ttacctagtg
     3361 gagacaaaag tacatacctc ggtagagcgc gcacgcctgt aaccccagct cgaaacctta
     3421 aggaatttcg agggggggcc cggtacccag cttttgttcc ctttagtgag ggttaattgc
     3481 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     3541 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     3601 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     3661 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     3721 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     3781 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     3841 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     3901 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3961 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     4021 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     4081 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     4141 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     4201 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     4261 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     4321 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     4381 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     4441 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     4501 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     4561 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     4621 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     4681 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     4741 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     4801 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     4861 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     4921 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4981 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     5041 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     5101 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     5161 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     5221 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     5281 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     5341 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     5401 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     5461 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     5521 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     5581 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     5641 agtgccac
//
