LOCUS       sequence-405470-        8611 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRegev_S4 with double mutations that restore secondary structure..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8611)
  AUTHORS   Liao SE, Sudarshan M, Regev O
  TITLE     Deciphering RNA splicing logic with interpretable machine learning.
  JOURNAL   Proc Natl Acad Sci U S A. 2023 Oct 10;120(41):e2221165120. doi: 
            10.1073/pnas.2221165120. Epub 2023 Oct 5.
  PUBMED    37796983
REFERENCE   2  (bases 1 to 8611)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2023-10-10"; volume: "120(41):e2221165120. 
            doi"; pages: " 10.1073/pnas.2221165120. Epub 2023 Oct 5"
COMMENT     Sequence Label: pRegev_S4.39U>G.51A>C
FEATURES             Location/Qualifiers
     source          1..8611
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(44..757,758..760,761..763))
                     /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(89..110)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     438..457
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     697..718
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      760..769
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(771..1949)
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          780..1718
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     complement(803..823)
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     promoter        1980..2257
                     /label=chicken beta-actin promoter
     primer_bind     3198..3220
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3247..3266
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3247..3265
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4631..4648)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4637..4861
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4907..5335
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4994..5013)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5204..5225
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5344..5364)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5349..5678
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5529..5664
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5591..5610
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5726..5773
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5792..6166
                     /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    6296..6417
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6333..6352)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6387..6406
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6466..6482)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6466..6482)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6479..6501)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6490..6506
                     /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(6514..6520,6521..6538,6539..6544))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6559..6580
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6697..6714)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6868..7456)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6948..6967)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7627..8418,8419..8487))
                     /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     8250..8269
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8488..8592)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccacctgacg tcgacggatc gggagatccc agtgtggtgg tacttacttg tacagctcgt
       61 ccatgccgag agtgatcccg gcggcggtca cgaactccag caggaccatg tgatcgcgct
      121 tctcgttggg gtctttgctc agggcggact gggtgctcag gtagtggttg tcgggcagca
      181 gcacggggcc gtcgccgatg ggggtgttct gctggtagtg gtcggcgagc tgcacgctgc
      241 cgtcctcgat gttgtggcgg atcttgaagt tcgccttgat gccgttcttc tgcttgtcgg
      301 ccatgatata gacgttgtgg ctgttgtagt tgtactccag cttgtgcccc aggatgttgc
      361 cgtcctcctt gaagtcgatg cccttcagct cgatgcggtt caccagggtg tcgccctcga
      421 acttcacctc ggcgcgggtc ttgtagttgc cgtcgtcctt gaagaagatg gtgcgctcct
      481 ggacgtagcc ttcgggcatg gcggacttga agaagtcgtg ctgcttcatg tggtcggggt
      541 agcggctgaa gcactgcacg ccgtaggtca gggtggtcac gagggtgggc cagggcacgg
      601 gcagcttgcc ggtggtgcag atgaacttca gggtcagctt gccgtaggtg gcatcgccct
      661 cgccctcgcc agacacgctg aacttgtggc cgtttacgtc gccgtccagc tcgaccagga
      721 tgggcaccac cccggtgaac agctcctcgc ccttgctcac catggtggcc tcacgacacc
      781 tgaaatggaa gaaaaaaact ttgaaccact gtctgaggct tgagaatgaa ccaagatcca
      841 aactcaaaaa gggcaaattc caaggagaat tacatcaagt gccaagctgg cctaacttca
      901 gtctccaccc actcagtgtg gggaaactcc atcgcataaa acccctcccc ccaacctaaa
      961 gacgacgtac tccaaaagct cgagaactaa tcgaggtgcc tggacggcgc ccggtactcc
     1021 gtggagtcac atgaagcgac ggctgaggac ggaaaggccc ttttcctttg tgtgggtgac
     1081 tcacccgccc gctctcccga gcgccgcgtc ctccattttg agctccctgc agcagggccg
     1141 ggaagcggcc atctttccgc tcacgcaact ggtgccgacc gggccagcct tgccgcccag
     1201 ggcggggcga tacacggcgg cgcgaggcca ggcaccagag caggccggcc agcttgagac
     1261 tacccccgtc cgattctcgg tggccgcgct cgcaggcccc gcctcgccga acatgtgcgc
     1321 tgggacgcac gggccccgtc gccgcccgcg gccccaaaaa ccgaaatacc agtgtgcaga
     1381 tcttggcccg catttacaag actatcttgc cagaaaaaaa gcgtcgcagc aggtcatcaa
     1441 aaattttaaa tggctagaga cttatcgaaa gcagcgagac aggcgcgaag gtgccaccag
     1501 attcgcacgc ggcggcccca gcgcccaggc caggcctcaa ctcaagcacg aggcgaaggg
     1561 gctccttaag cgcaaggcct cgaactctcc cacccacttc caacccgaag ctcgggatca
     1621 agaatcacgt actgcagcca ggtggaagta attcaaggca cgcaagggcc ataacccgta
     1681 aagaggccag gcccgcggga accacacacg gcacttacct gtgttctggc ggcaaacccg
     1741 ttgcgaaaaa gaacgttcac ggcgactact gcacttatat acggttctcc cccaccctcg
     1801 ggaaaaaggc ggagccagta cacgacatca ctttcccagt ttaccccgcg ccaccttctc
     1861 taggcaccgg ttcaattgcc gacccctccc cccaacttct cggggactgt gggcgatgtg
     1921 cgctctgccc actgacgggc accggagcct acgtaggaat taattcgcca gcacagtggt
     1981 cgaggtgagc cccacgttct gcttcactct ccccatctcc cccccctccc cacccccaat
     2041 tttgtattta tttatttttt aattattttg tgcagcgatg ggggcggggg gggggggggg
     2101 gcgcgcgcca ggcggggcgg ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc
     2161 ggcggcagcc aatcagagcg gcgcgctccg aaagtttcct tttatggcga ggcggcggcg
     2221 gcggcggccc tataaaaagc gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg
     2281 ccccgtgccc cgctccgccg ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt
     2341 actcccacag gtgagcgggc gggacggccc ttctcctccg ggctgtaatt agcgcttggt
     2401 ttaatgacgg cttgtttctt ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg
     2461 ccctttgtgc ggggggagcg gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc
     2521 cgcgtgcggc tccgcgctgc ccggcggctg tgagcgctgc gggcgcggcg cggggctttg
     2581 tgcgctccgc agtgtgcgcg aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg
     2641 gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg
     2701 tgtgggcgcg tcggtcgggc tgcaaccccc cctgcacccc cctccccgag ttgctgagca
     2761 cggcccggct tcgggtgcgg ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg
     2821 cggggggtgg cggcaggtgg gggtgccggg cggggcgggg ccgcctcggg ccggggaggg
     2881 ctcgggggag gggcgcggcg gcccccggag cgccggcggc tgtcgaggcg cggcgagccg
     2941 cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat
     3001 ctgtgcggag ccgaaatctg ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa
     3061 gcggtgcggc gccggcagga aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg
     3121 ccgtcccctt ctccctctcc agcctcgggg ctgtccgcgg ggggacggct gccttcgggg
     3181 gggacggggc agggcggggt tcggcttctg gcgtgtgacc ggcggctcta gcgtttaaac
     3241 ttaagctaat acgactcact atagggagct tggtaccgca acctcaaaca gacaccatgg
     3301 tgcacctgac tcctgaggag aagtctgccg ttactgccct gtggggcaag gtgaacgtgg
     3361 atgaagttgg tggtgaggcc ctgggcaggt tggtatcaag gttacaagac aggtttaagg
     3421 agaccaatag aaactgggca tatggagaca gagaagactc ttgggtttct gataggcact
     3481 gactctctct gcctatgtct ttctctgcca tccaggttag atcagaacac ccactacatc
     3541 atggccacgc gccgggcact acgcccgggt ctaaagacca atccattcca ggtctgacta
     3601 tgggaccctt gatgttttct ttccccttct tttctatggt taagttcatg tcataggaag
     3661 gggagaagta acagggtaca gtttagaatg ggaaacagac gaatgattgc atcagtgtgg
     3721 aagtctcagg atcgttttag tttcttttat ttgctgttca taacaattgt tttcttttgt
     3781 ttaattcttg ctttcttttt ttttcttctc cgcaattttt actattatac ttaatgcctt
     3841 aacattgtgt ataacaaaag gaaatatctc tgagatacat taagtaactt aaaaaaaaac
     3901 tttacacagt ctgcctagta cattactatt tggaatatat gtgtgcttat ttgcatattc
     3961 ataatctccc tactttattt tcttttattt ttaattgata cataatcatt atacatattt
     4021 atgggttaaa gtgtaatgtt ttaatatcga tacacatatt gaccaaatca gggtaatttt
     4081 gcatttgtaa ttttaaaaaa tgctttcttc ttttaatata cttttttgtt tatcttattt
     4141 ctaatacttt ccctaatctc tttctttcag ggcaataatg atacaatgta tcatgcctct
     4201 ttgcaccatt ctaaagaata acagtgataa tttctgggtt aaggcaatag caatatttct
     4261 gcatataaat atttctgcat ataaattgta actgatgtaa gaggtttcat attgctaata
     4321 gcagctacaa tccagctacc attctgcttt tattttatgg ttgggataag gctggattat
     4381 tctgagtcca agctaggccc ttttgctaat catgttcata cctcttatct tcctcccaca
     4441 gctcctgggc aacgtgctgg tctgtgtgct ggcccatcac tttggcaaag aattcacccc
     4501 accagtgcag gctgcctatc agaaagtggt ggctggtgtg gctaatgccc tggcccacaa
     4561 gtatcactaa gctcgctcta gactagacga ttaaattaca ttatagggcc cgtttaaacc
     4621 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     4681 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     4741 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     4801 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     4861 gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc
     4921 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     4981 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     5041 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     5101 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     5161 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5221 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     5281 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     5341 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta
     5401 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag
     5461 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa
     5521 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac
     5581 taattttttt tatttatgca gaggccgagg ccgcctctgc ctctgagcta ttccagaagt
     5641 agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctcccggga gcttgtatat
     5701 ccattttcgg atctgatcag cacgtgttga caattaatca tcggcatagt atatcggcat
     5761 agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg ccgttccggt
     5821 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
     5881 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
     5941 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
     6001 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
     6061 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
     6121 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tgctacgaga
     6181 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     6241 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     6301 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     6361 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     6421 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     6481 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     6541 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     6601 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     6661 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     6721 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     6781 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     6841 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     6901 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     6961 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     7021 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     7081 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     7141 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     7201 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     7261 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     7321 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     7381 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     7441 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     7501 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     7561 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     7621 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     7681 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     7741 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     7801 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     7861 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     7921 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     7981 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     8041 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     8101 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     8161 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     8221 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     8281 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     8341 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     8401 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     8461 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     8521 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     8581 aggggttccg cgcacatttc cccgaaaagt g
//
