LOCUS       nCre-ST-NLS-P2A-NLS-pSC 7041 bp ds-DNA     circular     02-AUG-2023
DEFINITION  .
COMMENT     Designed to mimic this paper's final construct:
            https://www.nature.com/articles/nchembio.2205#Sec18 Clone into
            pcDNA3.1+ with cutsites NheI and XbaI Can be the Hygro or Neo
            versions... depends what we want. I think the only difference
            between this and the SpyCatcher-GFP paper design is that the NLS
            for the N-terminal fragment is on the N-terminus. In my new design,
            I put a C-terminal NLS after SpyTag.The other thing the Nat Chem
            Bio paper found was that the length of the linker between Cre &
            your binding partner had an impact and that a shorter (2a.a.)
            linker performed best. This is probably binder protein dependent
            but I shortened my previous linker from 15a.a. to 6a.a. (we can
            change this back but something to be aware of). In previous designs
            I had included a second P2A sequence w/ BFP at the very end of the
            construct to track transfection/expression but that might have
            altered Cre's ability to properly fold around the DNA since the
            C-terminus would have most of the P2A sequence. I took that out for
            this design but we could try both?Finally, in past designs and
            here, the nCre-SpyTag fragment is first. This would allow for
            expression of more nCre-ST than SpyCatcher. Should we flip this?
            The only downfall is that the cCre fragment will contain that P2A
            scar of 21a.a.
FEATURES             Location/Qualifiers
     promoter        615..818
                     /label="CMV promoter"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     primer_bind     863..882
                     /label="T7"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     CDS             910..2427
                     /label="Translation 910-2427"
                     /translation="MATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNSGGSGSTAHIVMVDAYKPTKVPKKKRKVGSGATNFSLLKQAGDVEENPGPVPKKKRKVGSMVDTLSGLSSEQGQSGDMTIEEDSATHIKFS*RDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHISGGSGSTNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED"
     misc_feature    913..1038
                     /label="NCre19-59"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     misc_feature    1039..1059
                     /label="L1"
                     /ApEinfo_revcolor="#b1ff67"
                     /ApEinfo_fwdcolor="#b1ff67"
     misc_feature    1060..1098
                     /label="SpyTag"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     misc_feature    1099..1122
                     /label="NLS"
                     /ApEinfo_revcolor="#d6b295"
                     /ApEinfo_fwdcolor="#d6b295"
     Self-Cleaving   1123..1188
                     /label="P2A"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     misc_feature    1189..1212
                     /label="NLS"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     misc_feature    1219..1560
                     /label="photocaged SpyCatcher"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
     misc_feature    1561..1581
                     /label="L1"
                     /ApEinfo_revcolor="#ff9ccd"
                     /ApEinfo_fwdcolor="#ff9ccd"
     misc_feature    1582..2427
                     /label="CCre59"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
     CDS             2428..2430
                     /label="Translation 2428-2430"
                     /translation="*"
     rep_origin      2755..3061
                     /label="F1 ori"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     UniProt         3559..4581
                     /label="HygR"
                     /ApEinfo_revcolor="#f58a5e"
                     /ApEinfo_fwdcolor="#f58a5e"
     primer_bind     complement(4880..4900)
                     /label="M13-rev"
                     /ApEinfo_revcolor="#85dae9"
                     /ApEinfo_fwdcolor="#85dae9"
     misc_binding    complement(4906..4928)
                     /label="LacO"
                     /ApEinfo_revcolor="#84b0dc"
                     /ApEinfo_fwdcolor="#84b0dc"
     promoter        complement(4932..4962)
                     /label="lac promoter"
                     /ApEinfo_revcolor="#c7b0e3"
                     /ApEinfo_fwdcolor="#c7b0e3"
     rep_origin      complement(5268..5950)
                     /label="ColE1 origin"
                     /ApEinfo_revcolor="#9eafd2"
                     /ApEinfo_fwdcolor="#9eafd2"
     CDS             complement(6048..6707)
                     /label="AmpR"
                     /ApEinfo_revcolor="#ffef86"
                     /ApEinfo_fwdcolor="#ffef86"
ORIGIN
        1 GACGGATCGG GAGATCTCCC GATCCCCTAT GGTCGACTCT CAGTACAATC TGCTCTGATG
       61 CCGCATAGTT AAGCCAGTAT CTGCTCCCTG CTTGTGTGTT GGAGGTCGCT GAGTAGTGCG
      121 CGAGCAAAAT TTAAGCTACA ACAAGGCAAG GCTTGACCGA CAATTGCATG AAGAATCTGC
      181 TTAGGGTTAG GCGTTTTGCG CTGCTTCGCG ATGTACGGGC CAGATATACG CGTTGACATT
      241 GATTATTGAC TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA
      301 TGGAGTTCCG CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC
      361 CCCGCCCATT GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC
      421 ATTGACGTCA ATGGGTGGAC TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT
      481 ATCATATGCC AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT
      541 ATGCCCAGTA CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA
      601 TCGCTATTAC CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG
      661 ACTCACGGGG ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC
      721 AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG
      781 GTAGGCGTGT ACGGTGGGAG GTCTATATAA GCAGAGCTCT CTGGCTAACT AGAGAACCCA
      841 CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGC
      901 GCCGCCACCa tggccacctc tgatgaagtc aggaagaacc tgatggacat gttcagggac
      961 aggcaggcct tctctgaaca cacctggaag atgctcctgt ctgtgtgcag atcctgggct
     1021 gcctggtgca agctgaacAG CGGCGGCAGC GGGAGTACAg cacacatagt aatggtagac
     1081 gcctacaagc cgacgaagGT GCCCAAGAAG AAGAGGAAAG TCGGAAGCGG TGCCACCAAT
     1141 TTCTCCCTCC TGAAGCAGGC TGGGGACGTG GAGGAAAACC CTGGCCCCGT GCCCAAGAAG
     1201 AAGAGGAAAG TCggttccat ggttgatacc ttatcaggtt tatcaagtga gcaaggtcag
     1261 tccggtgata tgacaattga agaagatagt gctacccata ttaaattctc aTAGcgtgat
     1321 gaggacggca aagagttagc tggtgcaact atggagttgc gtgattcatc tggtaaaact
     1381 attagtacat ggatttcaga tggacaagtg aaagatttct acctgtatcc aggaaaatat
     1441 acatttgtcg aaaccgcagc accagacggt tatgaggtag caactgctat tacctttaca
     1501 gttaatgagc aaggtcaggt tactgtaaat ggcaaagcaa ctaaaggtga cgctcatatt
     1561 TCTGGTGGCA GTGGAAGCAC Taacaggaaa tggttccctg ctgaacctga ggatgtgagg
     1621 gactacctcc tgtacctgca agccagaggc ctggctgtga agaccatcca acagcacctg
     1681 ggccagctca acatgctgca caggagatct ggcctgcctc gcccttctga ctccaatgct
     1741 gtgtccctgg tgatgaggag aatcagaaag gagaatgtgg atgctgggga gagagccaag
     1801 caggccctgg cctttgaacg cactgacttt gaccaagtca gatccctgat ggagaactct
     1861 gacagatgcc aggacatcag gaacctggcc ttcctgggca ttgcctacaa caccctgctg
     1921 cgcattgccg aaattgccag aatcagagtg aaggacatct cccgcaccga tggtgggaga
     1981 atgctgatcc acattggcag gaccaagacc ctggtgtcca cagctggtgt ggagaaggcc
     2041 ctgtccctgg gggttaccaa gctggtggag agatggatct ctgtgtctgg tgtggctgat
     2101 gaccccaaca actacctgtt ctgccgggtc agaaagaatg gtgtggctgc cccttctgcc
     2161 acctcccaac tgtccacccg ggccctggaa gggatctttg aggccaccca ccgcctgatc
     2221 tatggtgcca aggatgactc tgggcagaga tacctggcct ggtctggcca ctctgccaga
     2281 gtgggtgctg ccagggacat ggccagggct ggtgtgtcca tccctgaaat catgcaggct
     2341 ggtggctgga ccaatgtgaa cattgtgatg aactacatca gaaacctgga ctctgagact
     2401 ggggccatgg tgaggctgct cgaggattaa TCTAGAGGGC CCGTTTAAAC CCGCTGATCA
     2461 GCCTCGACTG TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC
     2521 TTGACCCTGG AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG
     2581 CATTGTCTGA GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG
     2641 GAGGATTGGG AAGACAATAG CAGGCATGCT GGGGATGCGG TGGGCTCTAT GGCTTCTGAG
     2701 GCGGAAAGAA CCAGCTGGGG CTCTAGGGGG TATCCCCACG CGCCCTGTAG CGGCGCATTA
     2761 AGCGCGGCGG GTGTGGTGGT TACGCGCAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG
     2821 CCCGCTCCTT TCGCTTTCTT CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA
     2881 GCTCTAAATC GGGGCATCCC TTTAGGGTTC CGATTTAGTG CTTTACGGCA CCTCGACCCC
     2941 AAAAAACTTG ATTAGGGTGA TGGTTCACGT AGTGGGCCAT CGCCCTGATA GACGGTTTTT
     3001 CGCCCTTTGA CGTTGGAGTC CACGTTCTTT AATAGTGGAC TCTTGTTCCA AACTGGAACA
     3061 ACACTCAACC CTATCTCGGT CTATTCTTTT GATTTATAAG GGATTTTGGG GATTTCGGCC
     3121 TATTGGTTAA AAAATGAGCT GATTTAACAA AAATTTAACG CGAATTAATT CTGTGGAATG
     3181 TGTGTCAGTT AGGGTGTGGA AAGTCCCCAG GCTCCCCAGG CAGGCAGAAG TATGCAAAGC
     3241 ATGCATCTCA ATTAGTCAGC AACCAGGTGT GGAAAGTCCC CAGGCTCCCC AGCAGGCAGA
     3301 AGTATGCAAA GCATGCATCT CAATTAGTCA GCAACCATAG TCCCGCCCCT AACTCCGCCC
     3361 ATCCCGCCCC TAACTCCGCC CAGTTCCGCC CATTCTCCGC CCCATGGCTG ACTAATTTTT
     3421 TTTATTTATG CAGAGGCCGA GGCCGCCTCT GCCTCTGAGC TATTCCAGAA GTAGTGAGGA
     3481 GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA AAGCTCCCGG GAGCTTGTAT ATCCATTTTC
     3541 GGATCTGATC AGCACGTGAT GAAAAAGCCT GAACTCACCG CGACGTCTGT CGAGAAGTTT
     3601 CTGATCGAAA AGTTCGACAG CGTCTCCGAC CTGATGCAGC TCTCGGAGGG CGAAGAATCT
     3661 CGTGCTTTCA GCTTCGATGT AGGAGGGCGT GGATATGTCC TGCGGGTAAA TAGCTGCGCC
     3721 GATGGTTTCT ACAAAGATCG TTATGTTTAT CGGCACTTTG CATCGGCCGC GCTCCCGATT
     3781 CCGGAAGTGC TTGACATTGG GGAATTCAGC GAGAGCCTGA CCTATTGCAT CTCCCGCCGT
     3841 GCACAGGGTG TCACGTTGCA AGACCTGCCT GAAACCGAAC TGCCCGCTGT TCTGCAGCCG
     3901 GTCGCGGAGG CCATGGATGC GATCGCTGCG GCCGATCTTA GCCAGACGAG CGGGTTCGGC
     3961 CCATTCGGAC CGCAAGGAAT CGGTCAATAC ACTACATGGC GTGATTTCAT ATGCGCGATT
     4021 GCTGATCCCC ATGTGTATCA CTGGCAAACT GTGATGGACG ACACCGTCAG TGCGTCCGTC
     4081 GCGCAGGCTC TCGATGAGCT GATGCTTTGG GCCGAGGACT GCCCCGAAGT CCGGCACCTC
     4141 GTGCACGCGG ATTTCGGCTC CAACAATGTC CTGACGGACA ATGGCCGCAT AACAGCGGTC
     4201 ATTGACTGGA GCGAGGCGAT GTTCGGGGAT TCCCAATACG AGGTCGCCAA CATCTTCTTC
     4261 TGGAGGCCGT GGTTGGCTTG TATGGAGCAG CAGACGCGCT ACTTCGAGCG GAGGCATCCG
     4321 GAGCTTGCAG GATCGCCGCG GCTCCGGGCG TATATGCTCC GCATTGGTCT TGACCAACTC
     4381 TATCAGAGCT TGGTTGACGG CAATTTCGAT GATGCAGCTT GGGCGCAGGG TCGATGCGAC
     4441 GCAATCGTCC GATCCGGAGC CGGGACTGTC GGGCGTACAC AAATCGCCCG CAGAAGCGCG
     4501 GCCGTCTGGA CCGATGGCTG TGTAGAAGTA CTCGCCGATA GTGGAAACCG ACGCCCCAGC
     4561 ACTCGTCCGA GGGCAAAGGA ATAGCACGTG CTACGAGATT TCGATTCCAC CGCCGCCTTC
     4621 TATGAAAGGT TGGGCTTCGG AATCGTTTTC CGGGACGCCG GCTGGATGAT CCTCCAGCGC
     4681 GGGGATCTCA TGCTGGAGTT CTTCGCCCAC CCCAACTTGT TTATTGCAGC TTATAATGGT
     4741 TACAAATAAA GCAATAGCAT CACAAATTTC ACAAATAAAG CATTTTTTTC ACTGCATTCT
     4801 AGTTGTGGTT TGTCCAAACT CATCAATGTA TCTTATCATG TCTGTATACC GTCGACCTCT
     4861 AGCTAGAGCT TGGCGTAATC ATGGTCATAG CTGTTTCCTG TGTGAAATTG TTATCCGCTC
     4921 ACAATTCCAC ACAACATACG AGCCGGAAGC ATAAAGTGTA AAGCCTGGGG TGCCTAATGA
     4981 GTGAGCTAAC TCACATTAAT TGCGTTGCGC TCACTGCCCG CTTTCCAGTC GGGAAACCTG
     5041 TCGTGCCAGC TGCATTAATG AATCGGCCAA CGCGCGGGGA GAGGCGGTTT GCGTATTGGG
     5101 CGCTCTTCCG CTTCCTCGCT CACTGACTCG CTGCGCTCGG TCGTTCGGCT GCGGCGAGCG
     5161 GTATCAGCTC ACTCAAAGGC GGTAATACGG TTATCCACAG AATCAGGGGA TAACGCAGGA
     5221 AAGAACATGT GAGCAAAAGG CCAGCAAAAG GCCAGGAACC GTAAAAAGGC CGCGTTGCTG
     5281 GCGTTTTTCC ATAGGCTCCG CCCCCCTGAC GAGCATCACA AAAATCGACG CTCAAGTCAG
     5341 AGGTGGCGAA ACCCGACAGG ACTATAAAGA TACCAGGCGT TTCCCCCTGG AAGCTCCCTC
     5401 GTGCGCTCTC CTGTTCCGAC CCTGCCGCTT ACCGGATACC TGTCCGCCTT TCTCCCTTCG
     5461 GGAAGCGTGG CGCTTTCTCA ATGCTCACGC TGTAGGTATC TCAGTTCGGT GTAGGTCGTT
     5521 CGCTCCAAGC TGGGCTGTGT GCACGAACCC CCCGTTCAGC CCGACCGCTG CGCCTTATCC
     5581 GGTAACTATC GTCTTGAGTC CAACCCGGTA AGACACGACT TATCGCCACT GGCAGCAGCC
     5641 ACTGGTAACA GGATTAGCAG AGCGAGGTAT GTAGGCGGTG CTACAGAGTT CTTGAAGTGG
     5701 TGGCCTAACT ACGGCTACAC TAGAAGGACA GTATTTGGTA TCTGCGCTCT GCTGAAGCCA
     5761 GTTACCTTCG GAAAAAGAGT TGGTAGCTCT TGATCCGGCA AACAAACCAC CGCTGGTAGC
     5821 GGTGGTTTTT TTGTTTGCAA GCAGCAGATT ACGCGCAGAA AAAAAGGATC TCAAGAAGAT
     5881 CCTTTGATCT TTTCTACGGG GTCTGACGCT CAGTGGAACG AAAACTCACG TTAAGGGATT
     5941 TTGGTCATGA GATTATCAAA AAGGATCTTC ACCTAGATCC TTTTAAATTA AAAATGAAGT
     6001 TTTAAATCAA TCTAAAGTAT ATATGAGTAA ACTTGGTCTG ACAGTTACCA ATGCTTAATC
     6061 AGTGAGGCAC CTATCTCAGC GATCTGTCTA TTTCGTTCAT CCATAGTTGC CTGACTCCCC
     6121 GTCGTGTAGA TAACTACGAT ACGGGAGGGC TTACCATCTG GCCCCAGTGC TGCAATGATA
     6181 CCGCGAGACC CACGCTCACC GGCTCCAGAT TTATCAGCAA TAAACCAGCC AGCCGGAAGG
     6241 GCCGAGCGCA GAAGTGGTCC TGCAACTTTA TCCGCCTCCA TCCAGTCTAT TAATTGTTGC
     6301 CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT AATAGTTTGC GCAACGTTGT TGCCATTGCT
     6361 ACAGGCATCG TGGTGTCACG CTCGTCGTTT GGTATGGCTT CATTCAGCTC CGGTTCCCAA
     6421 CGATCAAGGC GAGTTACATG ATCCCCCATG TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT
     6481 CCTCCGATCG TTGTCAGAAG TAAGTTGGCC GCAGTGTTAT CACTCATGGT TATGGCAGCA
     6541 CTGCATAATT CTCTTACTGT CATGCCATCC GTAAGATGCT TTTCTGTGAC TGGTGAGTAC
     6601 TCAACCAAGT CATTCTGAGA ATAGTGTATG CGGCGACCGA GTTGCTCTTG CCCGGCGTCA
     6661 ATACGGGATA ATACCGCGCC ACATAGCAGA ACTTTAAAAG TGCTCATCAT TGGAAAACGT
     6721 TCTTCGGGGC GAAAACTCTC AAGGATCTTA CCGCTGTTGA GATCCAGTTC GATGTAACCC
     6781 ACTCGTGCAC CCAACTGATC TTCAGCATCT TTTACTTTCA CCAGCGTTTC TGGGTGAGCA
     6841 AAAACAGGAA GGCAAAATGC CGCAAAAAAG GGAATAAGGG CGACACGGAA ATGTTGAATA
     6901 CTCATACTCT TCCTTTTTCA ATATTATTGA AGCATTTATC AGGGTTATTG TCTCATGAGC
     6961 GGATACATAT TTGAATGTAT TTAGAAAAAT AAACAAATAG GGGTTCCGCG CACATTTCCC
     7021 CGAAAAGTGC CACCTGACGT C
//