LOCUS       sequence-156000-        5171 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 5171)
  AUTHORS   Abbate J, Lacayo JC, Prichard M, Pari G, McVoy MA
  TITLE     Bifunctional protein conferring enhanced green fluorescence and 
            puromycin resistance.
  JOURNAL   Biotechniques. 2001 Aug;31(2):336-40.
  PUBMED    11515370
REFERENCE   2  (bases 1 to 5171)
  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: 
            "Biotechniques."; date: "2001-08"; volume: "31(2)"; pages: "336-40"
COMMENT     Sequence Label: pEGFP-puro
FEATURES             Location/Qualifiers
     source          1..5171
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     24..40
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     24..40
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     enhancer        114..417
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        418..621
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     572..592
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      660..669
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(666..668,669..671,672..1382)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(666..668,669..671,672..1382)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(711..732)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(972..991)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1319..1340
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1413..2009
                     /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(1413..1432)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1909..1929
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     polyA_signal    2139..2260
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2176..2195)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2230..2249
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2277..2293)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2278..2294)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(2318..2337)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2319..2337)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2344..2361)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2344..2360)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2353..2375)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2505..2960)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2642..2663)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2854..2873
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2989..3093
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3094..3162,3163..3954)
                     /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(3312..3331)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4125..4713
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4614..4633
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4867..4884
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5001..5022
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5037..5042,5043..5060,5061..5067)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5075..5091
                     /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)."
     primer_bind     5080..5102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5099..5115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5099..5115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5136..5154
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 agctccaccg cggtggcggc cgctctagaa ctagtggatc ccccgggctg caggaattcg
       61 atatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
      121 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      181 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      241 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      301 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      361 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      421 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      481 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      541 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
      601 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg
      661 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
      721 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
      781 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
      841 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
      901 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
      961 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     1021 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1081 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     1141 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     1201 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1261 accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca
     1321 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     1381 agtccggact cagatctagg agacgacctt ccatgaccga gtacaagccc acggtgcgcc
     1441 tcgccacccg cgacgacgtc ccccgggccg tacgcaccct cgccgccgcg ttcgccgact
     1501 accccgccac gcgccacacc gtcgacccgg accgccacat cgagcgggtc accgagctgc
     1561 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     1621 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     1681 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     1741 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     1801 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     1861 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     1921 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     1981 cctggtgcat gacccgcaag cccggtgcgg gatccaccgg atctagataa ctgatcataa
     2041 tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc
     2101 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     2161 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     2221 attctagttg tggtttgtcc aaactcatca atgtatctta acgcgatcaa gcttatcgat
     2281 accgtcgacc tcgagggggg gcccggtacc caattcgccc tatagtgagt cgtattacaa
     2341 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     2401 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     2461 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg cgcgaaattg taaacgttaa
     2521 tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc
     2581 cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt
     2641 tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa
     2701 aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg
     2761 gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg
     2821 acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc
     2881 tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa
     2941 tgcgccgcta cagggcgcgt cccaggtggc acttttcggg gaaatgtgcg cggaacccct
     3001 atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga
     3061 taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc
     3121 cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg
     3181 aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc
     3241 aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact
     3301 tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca agagcaactc
     3361 ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag
     3421 catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat
     3481 aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt
     3541 ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa
     3601 gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac aacgttgcgc
     3661 aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg
     3721 gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt
     3781 gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca
     3841 gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat
     3901 gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca
     3961 gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg
     4021 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4081 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4141 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4201 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4261 ccaaatactg tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4321 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4381 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4441 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4501 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4561 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4621 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     4681 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     4741 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     4801 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     4861 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     4921 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     4981 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     5041 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     5101 ggaaacagct atgaccatga ttacgccaag ctcggaatta accctcacta aagggaacaa
     5161 aagctggagc t
//
