LOCUS       sequence-408322-        5654 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses ALFA-tagged MCP fusion protein in mammalian cells (with a 
            CMV promoter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5654)
  AUTHORS   Choi J, Chen W, Liao H, Li X, Shendure J
  TITLE     A molecular proximity sensor based on an engineered, dual-component 
            guide RNA.
  JOURNAL   Elife. 2025 Feb 12;13:RP98110. doi: 10.7554/eLife.98110.
  PUBMED    39937081
REFERENCE   2  (bases 1 to 5654)
  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: "2025-02-12"; volume: "13:RP98110. doi"; pages: " 
            10.7554/eLife.98110"
COMMENT     Sequence Label: pCMV-ALFA-MCP
FEATURES             Location/Qualifiers
     source          1..5654
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1440..1643
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1594..1614
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1640..1664
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     1719..1738
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1719..1737
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1745..1754
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1763..1801
                     /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"
     polyA_signal    2224..2345
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2261..2280)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2315..2334
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   3108..3142
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(3633..3649)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3633..3649)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3646..3668)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3657..3673
                     /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(3681..3687,3688..3705,3706..3711))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3726..3747
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3864..3881)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4035..4623)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4115..4134)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4794..5585,5586..5654))
                     /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     5417..5436
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggccacta gtattatgcc cagtacatga
     1381 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     1441 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc
     1501 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1561 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt
     1621 gggaggttta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg agacgccatc
     1681 cacgctgttt tgacctccat agaagattct agagctagta atacgactca ctatagggag
     1741 agccgccacc atgggaggac ccagcagact ggaagaggaa ctgagacgga gactgaccga
     1801 gcctggctct ggcggctcag gaagcggagc cagcaatttc acccagttcg tgctggtcga
     1861 caacggcggt acaggcgatg tgaccgtggc ccctagcaac ttcgccaacg gcgtggccga
     1921 gtggatcagc agcaacagca gaagccaggc ctataaggtg acatgcagcg tgcggcagtc
     1981 ttctgcccag aagcgcaagt acaccatcaa ggtggaggtg cctaaagtgg ctacccaaac
     2041 agtgggaggc gtggagctgc ctgtggcagc ctggcggagc tacctgaaca tggaactcac
     2101 catccctatc ttcgccacga acagcgactg tgagctgatc gtgaaagcca tgcagggcct
     2161 gctgaaggac ggcaacccca tcccttctgc catcgccgct aatagtggac tgtactaaaa
     2221 ctaaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     2281 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     2341 tcttatcatg tctggaattg actcaaatga tgtcaattag tctatcagaa gctcatctgg
     2401 tctcccttcc gggggacaag acatccctgt ttaatattta aacagcagtg ttcccaaact
     2461 gggttcttat atcccttgct ctggtcaacc aggttgcagg gtttcctgtc ctcacaggaa
     2521 cgaagtccct aaagaaacag tggcagccag gtttagcccc ggaattgact ggattccttt
     2581 tttagggccc attggtatgg ctttttcccc gtatcccccc aggtgtctgc aggctcaaag
     2641 agcagcgaga agcgttcaga ggaaagcgat cccgtgccac cttccccgtg cccgggctgt
     2701 ccccgcacgc tgccggctcg gggatgcggg gggagcgccg gaccggagcg gagccccggg
     2761 cggctcgctg ctgcccccta gcgggggagg gacgtaatta catccctggg ggctttgggg
     2821 gggggctgtc cctgatatct ataacaagaa aatatatata taataagtta tcacgtaagt
     2881 agaacatgaa ataacaatat aattatcgta tgagttaaat cttaaaagtc acgtaaaaga
     2941 taatcatgcg tcattttgac tcacgcggtc gttatagttc aaaatcagtg acacttaccg
     3001 cattgacaag cacgcctcac gggagctcca agcggcgact gagatgtcct aaatgcacag
     3061 cgacggattc gcgctattta gaaagagaga gcaatatttc aagaatgcat gcgtcaattt
     3121 tacgcagact atctttctag ggttaatcta gctgcatcag gatcatatcg tcgggtcttt
     3181 tttccggctc agtcatcgcc caagctggcg ctatctgggc atcggggagg aagaagcccg
     3241 tgccttttcc cgcgaggttg aagcggcatg gaaagagttt gccgaggatg actgctgctg
     3301 cattgacgtt gagcgaaaac gcacgtttac catgatgatt cgggaaggtg tggccatgca
     3361 cgcctttaac ggtgaactgt tcgttcaggc cacctgggat accagttcgt cgcggctttt
     3421 ccggacacag ttccggatgg tcagcccgaa gcgcatcagc aacccgaaca ataccggcga
     3481 cagccggaac tgccgtgccg gtgtgcagat taatgacagc ggtgcggcgc tgggatatta
     3541 cgtcagcgag gacgggtatc ctggctggat gccgcagaaa tggacatgga taccccgtga
     3601 gttacccggc gggcgcgctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     3661 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     3721 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     3781 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     3841 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     3901 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     3961 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4021 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4081 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4141 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4201 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4261 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4321 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4381 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4441 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4501 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4561 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     4621 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     4681 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     4741 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     4801 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     4861 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     4921 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     4981 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5041 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5101 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5161 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5221 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5281 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5341 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5401 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5461 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5521 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     5581 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     5641 tgttgaatac tcat
//
