LOCUS       sequence-362008-        5894 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To express FLAG tagged eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5894)
  AUTHORS   Beucher A, Miguel-Escalada I, Balboa D, De Vas MG, Maestro MA, 
            Garcia-Hurtado J, Bernal A, Gonzalez-Franco R, Vargiu P, Heyn H, 
            Ravassard P, Ortega S, Ferrer J
  TITLE     The HASTER lncRNA promoter is a cis-acting transcriptional 
            stabilizer of HNF1A.
  JOURNAL   Nat Cell Biol. 2022 Oct 6. pii: 10.1038/s41556-022-00996-8. doi: 
            10.1038/s41556-022-00996-8.
  PUBMED    36202974
REFERENCE   2  (bases 1 to 5894)
  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: "Nat Cell 
            Biol. 2022 Oct 6. pii: 10.1038/s41556-022-00996-8. doi: 
            10.1038/s41556-022-00996-8."
COMMENT     Sequence Label: pcDNA3.1-GFP-Flag-UTR
FEATURES             Location/Qualifiers
     source          1..5894
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        183..562
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        563..766
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     717..737
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     811..830
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        811..829
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             join(906..908,909..1619)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(906..908,909..1619)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(948..969)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1209..1228)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1556..1577
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1632..1655
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     3'UTR           1659..1784
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(1850..1867)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1856..2080
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2126..2554
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2213..2232)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2423..2444
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2563..2583)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2568..2897
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2748..2883
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2810..2829
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        2945..2992
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3011..3385
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     polyA_signal    3515..3636
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3552..3571)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3606..3625
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3685..3701)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3685..3701)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3698..3720)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3709..3725
                     /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(3733..3739,3740..3757,3758..3763))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3778..3799
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3916..3933)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4087..4675)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4167..4186)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4846..5637,5638..5706))
                     /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     5469..5488
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5707..5811)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(join(5886..5894,1..11))
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg aggtcgctga
       61 gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca attgcatgaa
      121 gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca gatatacgcg
      181 ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
      241 cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc
      301 caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg
      361 gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca
      421 tcaagtgtat catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc
      481 ctggcattat gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
      541 attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg ggcgtggata
      601 gcggtttgac tcacggggat ttccaagtct ccaccccatt gacgtcaatg ggagtttgtt
      661 ttggcaccaa aatcaacggg actttccaaa atgtcgtaac aactccgccc cattgacgca
      721 aatgggcggt aggcgtgtac ggtgggaggt ctatataagc agagctctct ggctaactag
      781 agaacccact gcttactggc ttatcgaaat taatacgact cactataggg agacccaagc
      841 tggctagcgt ttaaacttaa gcttggtacc gagctcggat ccactagtcc agtgtggtgg
      901 gcaccatgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg
      961 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct
     1021 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
     1081 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
     1141 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
     1201 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
     1261 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
     1321 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
     1381 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
     1441 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
     1501 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg
     1561 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagg
     1621 aattcactag tgactacaaa gacgatgacg ataaataaaa ccagcctcaa gaacacccga
     1681 atggagtctc taagctacat aataccaact tacactttac aaaatgttgt cccccaaaat
     1741 gtagccattc gtatctgctc ctaataaaaa gaaagtttct tcacctgcag atatccagca
     1801 cagtggcggc cgctcgagtc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg
     1861 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1921 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1981 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2041 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2101 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2161 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2221 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     2281 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     2341 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     2401 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     2461 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     2521 aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag
     2581 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     2641 agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca
     2701 tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa
     2761 ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag
     2821 aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag
     2881 gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc
     2941 acgtgttgac aattaatcat cggcatagta tatcggcata gtataatacg acaaggtgag
     3001 gaactaaacc atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc
     3061 cggagcggtc gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga
     3121 cttcgccggt gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt
     3181 ggtgccggac aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga
     3241 gtggtcggag gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat
     3301 cggcgagcag ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca
     3361 cttcgtggcc gaggagcagg actgacacgt gctacgagat ttcgattcca ccgccgcctt
     3421 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3481 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     3541 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3601 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     3661 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     3721 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     3781 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3841 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3901 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3961 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     4021 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     4081 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     4141 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4201 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4261 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4321 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4381 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4441 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4501 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4561 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4621 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4681 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4741 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4801 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4861 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4921 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4981 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     5041 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     5101 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     5161 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5221 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5281 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5341 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5401 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5461 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5521 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5581 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5641 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5701 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5761 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5821 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
     5881 ctcagtacaa tctg
//
