LOCUS       sequence-14322-e        7253 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 7253)
  TITLE     mammalian homologous recombination reporter genes
REFERENCE   2  (bases 1 to 7253)
  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"
COMMENT     Sequence Label: pGCGFP-blcd
FEATURES             Location/Qualifiers
     source          1..7253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1678..1697
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1755..1775
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1797..1817
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      2031..2040
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2037..2039,2040..2042,2043..2756)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSG*G*QGNTYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     2690..2711
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2879..3000
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        complement(3444..3941)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3492..3511)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     3883..3906
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     CDS             complement(join(3991..4449,4450..4452,4453..4455))
                     /codon_start=1
                     /product="improved N-terminal fragment of mVenus for use in
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN155(I152L)
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMA"
     regulatory      4452..4461
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(4675..4695)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(4717..4737)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4771..4787)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4771..4787)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4784..4806)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4795..4811
                     /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(4819..4825,4826..4843,4844..4849))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4864..4885
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    5130..5264
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5332..5349)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5503..6091)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5583..5602)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6262..7053,7054..7122))
                     /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     6885..6904
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7123..7227)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtccgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttac gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcagatcc gccgccacta
     1741 tgggatcaag atcgccaaaa aagaagagaa aggtgccgaa gaagcatgca gcaccaccaa
     1801 aaaaaaaacg aaaagtagaa gacccacgag gcaacaccag cggcgtgctg agcaccccca
     1861 aggccaagag ggccaagcac ccccccggca ccgagaagcc caggagcagg agccagagcg
     1921 agcagcccgc cacctgcccc atctgctacg ccgtgatcag gcagagcagg aacctgagga
     1981 ggcacctgga gctgaggcac ttcgccaagc ccggcgtgga tccaccggtc gccaccatgg
     2041 tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg
     2101 acgtaaacgg ccacaagttc agcgtgtccg gctagggata acagggtaat acctacggca
     2161 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg
     2221 tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc
     2281 acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca
     2341 aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga
     2401 accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc
     2461 tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     2521 tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc
     2581 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     2641 tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     2701 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtaaagcg
     2761 gccgcgactc tagatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     2821 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     2881 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     2941 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     3001 agttagttcc tggtgtactt gagggggatg agttcctcaa tggtggtttt gaccagcttg
     3061 ccattcatct caatgagcac aaagcagtca ggagcatagt cagagatgag ctctctgcac
     3121 atgccacagg ggctgaccac cctgatggat ctgtccacct catcagagta ggggtgcctg
     3181 acagccacaa tggtgtcaaa gtccttctgc ccgttgctca cagcagaccc aatggcaatg
     3241 gcttcagcac agacagtgac cctgccaatg taggcctcaa tgtggacagc agagatgatc
     3301 tccccagtct tggtcctgat ggccgccccg acatggtgct tgttgtcctc atagagcatg
     3361 gtgatcttct cagtggcgac ctccaccagc tccagatcct gctgagagat gttgaaggtt
     3421 ttcatggtgg cggctctcga ggtcgaaagg cccggagatg aggaagagga gaacagcgcg
     3481 gcagacgtgc gcttttgaag cgtgcagaat gccgggcctc cggaggacct tcgggcgccc
     3541 gccccgcccc tgagcccgcc cctgagcccg cccccggacc caccccttcc cagcctctga
     3601 gcccagaaag cgaaggagca aagctgctat tggccgctgc cccaaaggcc tacccgcttc
     3661 cattgctcag cggtgctgtc catctgcacg agactagtga gacgtgctac ttccatttgt
     3721 cacgtcctgc acgacgcgag ctgcggggcg ggggggaact tcctgactag gggaggagta
     3781 gaaggtggcg cgaaggggcc accaaagaac ggagccggtt ggcgcctacc ggtggatgtg
     3841 gaatgtgtga ggccagaggc cacttgtgta gcgccaagtg cccagcgggg ctgctaaagc
     3901 gcatgctcca gactgccttg ggaaaagcgc ctcccctacc cggtagaagc ttttattgaa
     3961 gttcaccttg atgccgttct tctgcttgtc ggccatgata tagacgttgt ggctgttgta
     4021 gttgtactcc agcttgtgcc ccaggatgtt gccgtcctcc ttgaagtcga tgcccttcag
     4081 ctcgatgcgg ttcaccaggg tgtcgccctc gaacttcacc tcggcgcggg tcttgtagtt
     4141 gccgtcgtcc ttgaagaaga tggtgcgctc ctggacgtag ccttcgggca tggcggactt
     4201 gaagaagtcg tgctgcttca tgtggtcggg gtagcggctg aagcactgca cgccgtaggt
     4261 cagggtggtc acgagggtgg gccagggcac gggcagcttg ccggtggtgc agatgaactt
     4321 cagggtcagc ttgccgtagg tggcatcgcc ctcgccctcg ccggacacgc tgaacttgtg
     4381 gccgtttacg tcgccgtcca gctcgaccag gatgggcacc accccggtga acagctcctc
     4441 gcccttgctc accatggtgg cgaccggtgg atccacgccg ggcttggcga agtgcctcag
     4501 ctccaggtgc ctcctcaggt tcctgctctg cctgatcacg gcgtagcaga tggggcaggt
     4561 ggcgggctgc tcgctctggc tcctgctcct gggcttctcg gtgccggggg ggtgcttggc
     4621 cctcttggcc ttgggggtgc tcagcacgcc gctggtgttg cctcgtgggt cttctacttt
     4681 tcgttttttt tttggtggtg ctgcatgctt cttcggcacc tttctcttct tttttggcga
     4741 tcttgatccc taaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4801 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4861 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4921 aaacctgtcg tgccagcgga tccgcatctc aattagtcag caaccatagt cccgccccta
     4981 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     5041 ctaatttttt ttatttatgt ctccaacgcg tagctattcc agaagtagtg aggaggcttt
     5101 tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca
     5161 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     5221 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat
     5281 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5341 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5401 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5461 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5521 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5581 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5641 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaaag
     5701 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5761 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5821 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5881 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5941 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6001 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6061 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     6121 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     6181 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     6241 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     6301 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     6361 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     6421 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6481 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6541 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6601 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6661 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6721 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6781 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6841 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6901 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6961 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     7021 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     7081 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     7141 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     7201 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctg
//
