LOCUS       sequence-14327-e        7625 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 7625)
  TITLE     mammalian homologous recombination reporter genes
REFERENCE   2  (bases 1 to 7625)
  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-G418
FEATURES             Location/Qualifiers
     source          1..7625
                     /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"
     CDS             complement(3003..3797)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             complement(3003..3797)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3114..3133)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3724..3743
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(3816..4313)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3864..3883)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     4255..4278
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     CDS             complement(join(4363..4821,4822..4824,4825..4827))
                     /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      4824..4833
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(5047..5067)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             complement(5089..5109)
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(5143..5159)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5143..5159)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5156..5178)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5167..5183
                     /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(5191..5197,5198..5215,5216..5221))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5236..5257
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     polyA_signal    5502..5636
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5704..5721)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5875..6463)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5955..5974)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6634..7425,7426..7494))
                     /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     7257..7276
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7495..7599)
                     /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 agtcagaaga actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg
     3061 ataccgtaaa gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca
     3121 cgggtagcca acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg
     3181 aatccagaaa agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc
     3241 acgacgagat cctcgccgtc gggcatgctc gccttgagcc tggcgaacag ttcggctggc
     3301 gcgagcccct gatgctcttc gtccagatca tcctgatcga caagaccggc ttccatccga
     3361 gtacgtgctc gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca
     3421 agcgtatgca gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg
     3481 tgagatgaca ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct
     3541 tcagtgacaa cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc
     3601 cgcgctgcct cgtcttgcag ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga
     3661 accgggcgcc cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt
     3721 tgtgcccagt catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat
     3781 ccatcttgtt caatcatggt ggcggctctc gaggtcgaaa ggcccggaga tgaggaagag
     3841 gagaacagcg cggcagacgt gcgcttttga agcgtgcaga atgccgggcc tccggaggac
     3901 cttcgggcgc ccgccccgcc cctgagcccg cccctgagcc cgcccccgga cccacccctt
     3961 cccagcctct gagcccagaa agcgaaggag caaagctgct attggccgct gccccaaagg
     4021 cctacccgct tccattgctc agcggtgctg tccatctgca cgagactagt gagacgtgct
     4081 acttccattt gtcacgtcct gcacgacgcg agctgcgggg cgggggggaa cttcctgact
     4141 aggggaggag tagaaggtgg cgcgaagggg ccaccaaaga acggagccgg ttggcgccta
     4201 ccggtggatg tggaatgtgt gaggccagag gccacttgtg tagcgccaag tgcccagcgg
     4261 ggctgctaaa gcgcatgctc cagactgcct tgggaaaagc gcctccccta cccggtagaa
     4321 gcttttattg aagttcacct tgatgccgtt cttctgcttg tcggccatga tatagacgtt
     4381 gtggctgttg tagttgtact ccagcttgtg ccccaggatg ttgccgtcct ccttgaagtc
     4441 gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc tcgaacttca cctcggcgcg
     4501 ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc tcctggacgt agccttcggg
     4561 catggcggac ttgaagaagt cgtgctgctt catgtggtcg gggtagcggc tgaagcactg
     4621 cacgccgtag gtcagggtgg tcacgagggt gggccagggc acgggcagct tgccggtggt
     4681 gcagatgaac ttcagggtca gcttgccgta ggtggcatcg ccctcgccct cgccggacac
     4741 gctgaacttg tggccgttta cgtcgccgtc cagctcgacc aggatgggca ccaccccggt
     4801 gaacagctcc tcgcccttgc tcaccatggt ggcgaccggt ggatccacgc cgggcttggc
     4861 gaagtgcctc agctccaggt gcctcctcag gttcctgctc tgcctgatca cggcgtagca
     4921 gatggggcag gtggcgggct gctcgctctg gctcctgctc ctgggcttct cggtgccggg
     4981 ggggtgcttg gccctcttgg ccttgggggt gctcagcacg ccgctggtgt tgcctcgtgg
     5041 gtcttctact tttcgttttt tttttggtgg tgctgcatgc ttcttcggca cctttctctt
     5101 cttttttggc gatcttgatc cctaaagctt ggcgtaatca tggtcatagc tgtttcctgt
     5161 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5221 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5281 tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata
     5341 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     5401 ccccatggct gactaatttt ttttatttat gtctccaacg cgtagctatt ccagaagtag
     5461 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taacttgttt attgcagctt
     5521 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     5581 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgct
     5641 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     5701 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     5761 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     5821 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     5881 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     5941 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     6001 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     6061 gctttctcaa agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     6121 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     6181 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     6241 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     6301 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     6361 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     6421 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     6481 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     6541 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     6601 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     6661 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     6721 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     6781 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     6841 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     6901 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     6961 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     7021 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     7081 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     7141 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     7201 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     7261 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     7321 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     7381 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     7441 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     7501 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     7561 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     7621 acctg
//
