LOCUS       sequence-481555-        4611 bp    DNA     circular SYN 30-JUL-2025
DEFINITION  Express EGFP with an upstream T7 promoter in pUC19 backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4611)
  AUTHORS   Chen H, Liu S, Padula S, Lesman D, Griswold K, Lin A, Zhao T, 
            Marshall JL, Chen F
  TITLE     Efficient, continuous mutagenesis in human cells using a 
            pseudo-random DNA editor.
  JOURNAL   Nat Biotechnol. 2019 Dec 16. pii: 10.1038/s41587-019-0331-8. doi: 
            10.1038/s41587-019-0331-8.
  PUBMED    31844291
REFERENCE   2  (bases 1 to 4611)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 30, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2019 Dec 16. pii: 10.1038/s41587-019-0331-8. doi: 
            10.1038/s41587-019-0331-8."
COMMENT     Sequence Label: pUC19 - T7 pro - IRES - EGFP
FEATURES             Location/Qualifiers
     source          1..4611
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    139..707
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     misc_feature    157..709
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(301..318)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     528..547
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(721..723,724..726,727..1440)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     ANFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(766..787)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1027..1046)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1374..1395
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1561..1578)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1567..1791
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1924..1943)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     complement(1973..1989)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1973..1989)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1986..2008)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1997..2013
                     /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(2021..2027,2028..2045,2046..2051))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2066..2087
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2204..2221)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2375..2963)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2455..2474)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3134..3925,3926..3994))
                     /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     3757..3776
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3995..4099)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4167..4185
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(4223..4245)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     4345..4364
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4558..4580
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4572..4589
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     4573..4589
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 taatacgact cactataggg agacccaagc tggctagcgt ttaaacttaa gcttggtacc
       61 gagctcggat ccactagtcc agtgtggtgg aattctgcag atatccagca cagtggcggc
      121 cgctcgaggt taacgaattc cgcccccccc cccctaacgt tactggccga agccgcttgg
      181 aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca
      241 atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc
      301 ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag
      361 cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg
      421 gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac
      481 aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa
      541 gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc
      601 tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc
      661 ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat ggccacaacc
      721 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac
      781 ggcgacgtaa acggccacaa gttcagcgtg tctggcgagg gcgagggcga tgccacctac
      841 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc
      901 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag
      961 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc
     1021 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg
     1081 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac
     1141 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac
     1201 ggcatcaagg cgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc
     1261 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac
     1321 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
     1381 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa
     1441 tgaattaatt aagaattatc accgcttcta ttcagccagt aaggcctgtc tttaatggcc
     1501 tccggcatga gacactttct agagtcgagt ctagagggcc cgtttaaacc cgctgatcag
     1561 cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct
     1621 tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
     1681 attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg
     1741 aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg
     1801 cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa
     1861 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1921 ccgctccttt cgctttcttc ccttgcaggc atgcaagctt ggcgtaatca tggtcatagc
     1981 tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca
     2041 taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct
     2101 cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac
     2161 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2221 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2281 tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg
     2341 ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg
     2401 agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat
     2461 accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta
     2521 ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct
     2581 gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc
     2641 ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa
     2701 gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg
     2761 taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag
     2821 tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt
     2881 gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta
     2941 cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc
     3001 agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca
     3061 cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
     3121 cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat
     3181 ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct
     3241 taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt
     3301 tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat
     3361 ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta
     3421 atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
     3481 gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt
     3541 tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg
     3601 cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg
     3661 taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc
     3721 ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa
     3781 ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac
     3841 cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt
     3901 ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg
     3961 gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa
     4021 gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata
     4081 aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca
     4141 ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc
     4201 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt
     4261 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg
     4321 ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     4381 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc
     4441 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     4501 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     4561 agtcacgacg ttgtaaaacg acggccagtg aattcgagct cggtacccgg g
//
