LOCUS       sequence-398309-        8612 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  pRegev_S2 with double mutations that restore secondary structure..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8612)
  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 8612)
  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_S2.39U>G.51A>C
FEATURES             Location/Qualifiers
     source          1..8612
                     /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..5679
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5530..5665
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5592..5611
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        5727..5774
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5793..6167
                     /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    6297..6418
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6334..6353)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6388..6407
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6467..6483)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6467..6483)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6480..6502)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6491..6507
                     /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(6515..6521,6522..6539,6540..6545))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6560..6581
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6698..6715)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6869..7457)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6949..6968)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7628..8419,8420..8488))
                     /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     8251..8270
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8489..8593)
                     /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 ggaaaggtgc
     2161 ggcggcagcc aatcaaagcg 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 ctcgtttctt 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 tccaggtttc gctctctaaa aaagtcacct
     3541 acttaacacg acgggcctcc gtggcccgaa aataaaccac taagaaaaca 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 gggcatccct ttagggttcc gatttagtgc tttacggcac
     5101 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     5161 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     5221 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgggg
     5281 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc
     5341 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccaggc aggcagaagt
     5401 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca
     5461 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta
     5521 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     5581 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag
     5641 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata
     5701 tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatag tatatcggca
     5761 tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagt gccgttccgg
     5821 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct
     5881 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg accctgttca
     5941 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtg tgggtgcgcg
     6001 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttc cgggacgcct
     6061 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttc gccctgcgcg
     6121 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacac gtgctacgag
     6181 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     6241 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     6301 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     6361 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     6421 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     6481 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     6541 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     6601 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6661 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     6721 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     6781 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     6841 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     6901 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     6961 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     7021 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt
     7081 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     7141 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     7201 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     7261 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     7321 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     7381 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     7441 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     7501 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     7561 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     7621 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     7681 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     7741 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     7801 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     7861 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     7921 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     7981 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     8041 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     8101 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     8161 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     8221 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     8281 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     8341 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     8401 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     8461 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     8521 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     8581 taggggttcc gcgcacattt ccccgaaaag tg
//
