LOCUS       sequence-391141-        5780 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of the Gal4-ALFA-tag-VP16 synthetic transcription factor 
            containing the ALFA-tag sequence in mammalian cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5780)
  AUTHORS   Chernov KG, Manoilov KY, Oliinyk OS, Shcherbakova DM, Verkhusha VV
  TITLE     Photodegradable by Yellow-Orange Light degFusionRed Optogenetic 
            Module with Autocatalytically Formed Chromophore.
  JOURNAL   Int J Mol Sci. 2023 Mar 30;24(7):6526. doi: 10.3390/ijms24076526.
  PUBMED    37047499
REFERENCE   2  (bases 1 to 5780)
  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: "Int J Mol 
            Sci."; date: "2023-03-30"; volume: "24(7):6526. doi"; pages: " 
            10.3390/ijms24076526"
COMMENT     Sequence Label: pcDNA-ALFA-tag-Gal4-VP16
FEATURES             Location/Qualifiers
     source          1..5780
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(13..33)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        18..347
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      198..333
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     260..279
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        395..442
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             461..835
                     /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    965..1086
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1002..1021)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1056..1075
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1135..1151)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1135..1151)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1148..1170)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1159..1175
                     /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(1183..1189,1190..1207,1208..1213))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1228..1249
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1366..1383)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1537..2125)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1617..1636)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2296..3087,3088..3156))
                     /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     2919..2938
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3157..3261)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3336..3355)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        3527..3906
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        3907..4110
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4061..4081
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4155..4174
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4155..4173
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4212..4250
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             4284..4724
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             4746..4979
                     /codon_start=1
                     /gene="UL48"
                     /product="transcriptional activation domain of herpes 
                     simplex virus protein VP16 (Triezenberg et al., 1988; 
                     Cousens et al., 1989)"
                     /label=VP16 AD
                     /translation="APPTDVSLGDELHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGF
                     TPHDSAPYGALDMADFEFEQMFTDALGIDEYGG"
     CDS             4995..5024
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5040..5057
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5080..5097)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5086..5310
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(5356..5780,1..4)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5443..5462)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5653..5674
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
ORIGIN
        1 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
       61 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
      121 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
      181 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
      241 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
      301 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
      361 cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta
      421 tatcggcata gtataatacg acaaggtgag gaactaaacc atggccaagt tgaccagtgc
      481 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct
      541 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac
      601 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg
      661 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg
      721 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc
      781 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt
      841 gctacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt
      901 ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca
      961 ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     1021 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     1081 atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata
     1141 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     1201 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     1261 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1321 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     1381 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     1441 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     1501 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     1561 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     1621 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     1681 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     1741 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     1801 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     1861 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     1921 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     1981 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2041 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2101 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2161 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2221 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2281 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2341 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     2401 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     2461 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     2521 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     2581 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     2641 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     2701 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     2761 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     2821 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     2881 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     2941 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3001 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3061 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3121 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     3181 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3241 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
     3301 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
     3361 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
     3421 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
     3481 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
     3541 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     3601 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     3661 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     3721 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     3781 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     3841 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     3901 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     3961 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     4021 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     4081 gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac
     4141 tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta gttaagcttg
     4201 gtaccatgcc cagcagactg gaggaggagc tgagaagaag actgaccgag cccggaggtg
     4261 gtggatcagg aggaggcggc cgcatgaagc tactgtcttc tatcgaacaa gcatgcgata
     4321 tttgccgact taaaaagctc aagtgctcca aagaaaaacc gaagtgcgcc aagtgtctga
     4381 agaacaactg ggagtgtcgc tactctccca aaaccaaaag gtctccgctg actagggcac
     4441 atctgacaga agtggaatca aggctagaaa gactggaaca gctatttcta ctgatttttc
     4501 ctcgagaaga ccttgacatg attttgaaaa tggattcttt acaggatata aaagcattgt
     4561 taacaggatt atttgtacaa gataatgtga ataaagatgc cgtcacagat agattggctt
     4621 cagtggagac tgatatgcct ctaacattga gacagcatag aataagtgcg acatcatcat
     4681 cggaagagag tagtaacaaa ggtcaaagac agttgactgt atcgccggaa ttcccgggga
     4741 tctgggcccc cccgaccgat gtcagcctgg gggacgagct ccacttagac ggcgaggacg
     4801 tggcgatggc gcatgccgac gcgctagacg atttcgatct ggacatgttg ggggacgggg
     4861 attccccggg tccgggattt accccccacg actccgcccc ctacggcgct ctggatatgg
     4921 ccgacttcga gtttgagcag atgtttaccg atgcccttgg aattgacgag tacggtgggt
     4981 ctagagggcc cttcgaacaa aaactcatct cagaagagga tctgaatatg cataccggtc
     5041 atcatcacca tcaccattga gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt
     5101 gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc
     5161 ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt
     5221 ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca
     5281 ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct
     5341 ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     5401 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     5461 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     5521 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg
     5581 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     5641 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct
     5701 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     5761 tttaacaaaa atttaacgcg
//
