LOCUS       sequence-409880-        7040 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  A plasmid encoding N-terminal Cre fused to SpyTag (ST) and 
            photocaged SpyCatcher (pSC) fused to C-terminal Cre with a nuclear 
            localization signal (NLS) sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7040)
  AUTHORS   Ruskowitz ER, Munoz-Robles BG, Strange AC, Butcher CH, Kurniawan S, 
            Filteau JR, DeForest CA
  TITLE     Spatiotemporal functional assembly of split protein pairs through a 
            light-activated SpyLigation.
  JOURNAL   Nat Chem. 2023 May;15(5):694-704. doi: 10.1038/s41557-023-01152-x. 
            Epub 2023 Apr 17.
  PUBMED    37069270
REFERENCE   2  (bases 1 to 7040)
  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"; doi: 
            "10.1038/s41557-023-01152-x"; journalName: "Nat Chem"; date: 
            "2023-05"; volume: "15"; issue: "5"; pages: "694-704"
COMMENT     Sequence Label: nCre-ST-NLS-P2A-NLS-pSC-cCre
FEATURES             Location/Qualifiers
     source          1..7040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             151..189
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     CDS             193..213
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             223..279
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             283..303
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             313..651
                     /codon_start=1
                     /product="truncated version of the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyCatcher
                     /note="forms a covalent isopeptide bond with the SpyTag 
                     peptide (Zakeri et al., 2012)"
                     /translation="VDTLSGLSSEQGQSGDMTIEEDSATHIKFS*RDEDGKELAGATME
                     LRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     KATKGDAHI"
     primer_bind     complement(1553..1570)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1559..1783
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1829..2257
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1916..1935)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2126..2147
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2266..2286)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2271..2600
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2451..2586
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2513..2532
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2649..3674
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2649..3674
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     polyA_signal    3804..3925
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3841..3860)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3895..3914
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3974..3990)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3974..3990)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3987..4009)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3998..4014
                     /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(4022..4028,4029..4046,4047..4052))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4067..4088
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4205..4222)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4376..4964)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4456..4475)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5135..5926,5927..5995))
                     /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     5758..5777
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5996..6100)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6175..6194)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        6366..6745
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        6746..6949
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     6900..6920
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     6994..7013
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6994..7012
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      join(7035..7040,1..4)
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 atggccacct ctgatgaagt caggaagaac ctgatggaca tgttcaggga caggcaggcc
       61 ttctctgaac acacctggaa gatgctcctg tctgtgtgca gatcctgggc tgcctggtgc
      121 aagctgaaca gcggcggcag cgggagtaca gcacacatag taatggtaga cgcctacaag
      181 ccgacgaagg tgcccaagaa gaagaggaaa gtcggaagcg gtgccaccaa tttctccctc
      241 ctgaagcagg ctggggacgt ggaggaaaac cctggccccg tgcccaagaa gaagaggaaa
      301 gtcggttcca tggttgatac cttatcaggt ttatcaagtg agcaaggtca gtccggtgat
      361 atgacaattg aagaagatag tgctacccat attaaattct catagcgtga tgaggacggc
      421 aaagagttag ctggtgcaac tatggagttg cgtgattcat ctggtaaaac tattagtaca
      481 tggatttcag atggacaagt gaaagatttc tacctgtatc caggaaaata tacatttgtc
      541 gaaaccgcag caccagacgg ttatgaggta gcaactgcta ttacctttac agttaatgag
      601 caaggtcagg ttactgtaaa tggcaaagca actaaaggtg acgctcatat ttctggtggc
      661 agtggaagca ctaacaggaa atggttccct gctgaacctg aggatgtgag ggactacctc
      721 ctgtacctgc aagccagagg cctggctgtg aagaccatcc aacagcacct gggccagctc
      781 aacatgctgc acaggagatc tggcctgcct cgcccttctg actccaatgc tgtgtccctg
      841 gtgatgagga gaatcagaaa ggagaatgtg gatgctgggg agagagccaa gcaggccctg
      901 gcctttgaac gcactgactt tgaccaagtc agatccctga tggagaactc tgacagatgc
      961 caggacatca ggaacctggc cttcctgggc attgcctaca acaccctgct gcgcattgcc
     1021 gaaattgcca gaatcagagt gaaggacatc tcccgcaccg atggtgggag aatgctgatc
     1081 cacattggca ggaccaagac cctggtgtcc acagctggtg tggagaaggc cctgtccctg
     1141 ggggttacca agctggtgga gagatggatc tctgtgtctg gtgtggctga tgaccccaac
     1201 aactacctgt tctgccgggt cagaaagaat ggtgtggctg ccccttctgc cacctcccaa
     1261 ctgtccaccc gggccctgga agggatcttt gaggccaccc accgcctgat ctatggtgcc
     1321 aaggatgact ctgggcagag atacctggcc tggtctggcc actctgccag agtgggtgct
     1381 gccagggaca tggccagggc tggtgtgtcc atccctgaaa tcatgcaggc tggtggctgg
     1441 accaatgtga acattgtgat gaactacatc agaaacctgg actctgagac tggggccatg
     1501 gtgaggctgc tcgaggatta atctagaggg cccgtttaaa cccgctgatc agcctcgact
     1561 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     1621 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     1681 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     1741 gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga ggcggaaaga
     1801 accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt aagcgcggcg
     1861 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct
     1921 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat
     1981 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt
     2041 gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg
     2101 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac
     2161 cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
     2221 aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt
     2281 tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2341 attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa
     2401 gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc
     2461 taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg
     2521 cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga ggcttttttg
     2581 gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc ggatctgatc
     2641 agcacgtgat gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa
     2701 agttcgacag cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca
     2761 gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct
     2821 acaaagatcg ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc
     2881 ttgacattgg ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg
     2941 tcacgttgca agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg
     3001 ccatggatgc gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac
     3061 cgcaaggaat cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc
     3121 atgtgtatca ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc
     3181 tcgatgagct gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg
     3241 atttcggctc caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga
     3301 gcgaggcgat gttcggggat tcccaatacg aggtcgccaa catcttcttc tggaggccgt
     3361 ggttggcttg tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag
     3421 gatcgccgcg gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct
     3481 tggttgacgg caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc
     3541 gatccggagc cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga
     3601 ccgatggctg tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga
     3661 gggcaaagga atagcacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt
     3721 tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca
     3781 tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa
     3841 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3901 tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct
     3961 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     4021 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     4081 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     4141 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4201 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4261 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4321 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4381 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4441 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4501 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4561 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4621 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4681 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4741 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4801 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4861 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4921 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4981 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5041 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5101 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5161 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5221 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5281 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5341 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5401 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5461 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5521 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5581 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5641 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5701 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5761 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5821 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5881 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5941 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6001 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6061 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6121 cacctgacgt cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat
     6181 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     6241 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     6301 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac
     6361 gcgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca
     6421 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc
     6481 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat
     6541 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
     6601 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
     6661 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
     6721 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg
     6781 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt
     6841 gttttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac
     6901 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc tctggctaac
     6961 tagagaaccc actgcttact ggcttatcga aattaatacg actcactata gggagaccca
     7021 agctggctag cgccgccacc
//
