LOCUS       sequence-343586-        5843 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expressing GFP in human cell lines via transfection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5843)
  AUTHORS   Xiang K, Bartel DP
  TITLE     The molecular basis of coupling between poly(A)-tail length and 
            translational efficiency.
  JOURNAL   Elife. 2021 Jul 2;10. pii: 66493. doi: 10.7554/eLife.66493.
  PUBMED    34213414
REFERENCE   2  (bases 1 to 5843)
  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: "Elife."; 
            date: "2021-07-2"; volume: "10. pii: 66493. doi"; pages: " 
            10.7554/eLife.66493"
COMMENT     Sequence Label: C194
FEATURES             Location/Qualifiers
     source          1..5843
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(533..1324,1325..1393))
                     /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     1156..1175
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1394..1498)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1573..1592)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1764..2143
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2144..2347
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2298..2318
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2349..2367
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2370..2388
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2398..2422
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      2516..2525
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2522..2524,2525..2527,2528..3241)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2522..2524,2525..2527,2528..3238)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2567..2588)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2828..2847)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3175..3196
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3324..3341)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3330..3554
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3687..3706)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3838..3885
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3893..4915
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3893..4915
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5045..5166
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5082..5101)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5136..5155
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5215..5231)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5215..5231)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5228..5250)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5239..5255
                     /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(5263..5269,5270..5287,5288..5293))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5308..5329
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5446..5463)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(5617..5843,1..362))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5697..5716)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
       61 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
      121 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
      181 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
      241 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
      301 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
      361 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
      421 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
      481 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
      541 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
      601 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
      661 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
      721 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
      781 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
      841 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
      901 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
      961 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     1021 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     1081 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     1141 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     1201 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     1261 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     1321 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     1381 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     1441 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     1501 catttccccg aaaagtgcca cctgacgtcg acggatcggg agatctcccg atcccctatg
     1561 gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtatc tgctccctgc
     1621 ttgtgtgttg gaggtcgctg agtagtgcgc gagcaaaatt taagctacaa caaggcaagg
     1681 cttgaccgac aattgcatga agaatctgct tagggttagg cgttttgcgc tgcttcgcga
     1741 tgtacgggcc agatatacgc gttgacattg attattgact agttattaat agtaatcaat
     1801 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
     1861 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
     1921 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
     1981 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
     2041 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
     2101 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
     2161 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
     2221 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
     2281 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
     2341 cagagctctc cctatcagtg atagagatct ccctatcagt gatagagatc gtcgacgagc
     2401 tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag
     2461 aagacaccgg gaccgatcca gcctccggac tctagcgttt aaacttaagc ttgccgccac
     2521 catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga
     2581 cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta
     2641 cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac
     2701 cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa
     2761 gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt
     2821 cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct
     2881 ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca
     2941 caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa
     3001 cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc
     3061 cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca
     3121 ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt
     3181 cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta
     3241 gtccagtgtg gtggaattct gcagatatcc agcacagtgg cggccgctcg agtctagagg
     3301 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3361 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     3421 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     3481 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     3541 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     3601 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     3661 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     3721 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     3781 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa
     3841 gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc
     3901 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     3961 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     4021 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     4081 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     4141 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     4201 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     4261 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     4321 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     4381 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     4441 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     4501 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     4561 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     4621 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     4681 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     4741 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     4801 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     4861 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt
     4921 actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
     4981 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
     5041 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     5101 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     5161 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     5221 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     5281 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     5341 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     5401 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     5461 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     5521 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     5581 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     5641 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     5701 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     5761 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     5821 ctgtaggtat ctcagttcgg tgt
//
