LOCUS       Addgene_(i32)2_I        5559 bp DNA     circular SYN 13-JUL-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5559)
  AUTHORS   The Francis Crick Institute
  TITLE     Direct Submission
  JOURNAL   Exported Jul 13, 2023 from SnapGene 6.2.2
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5559
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        21..39
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             70..960
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     primer_bind     875..895
                     /label=Halo Fwd
     CDS             973..993
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             1015..1554
                     /label=(i32)2
     CDS             1561..1935
                     /label=Talin R3 IVVI
     CDS             1942..2481
                     /label=(i32)2
     misc_feature    2488..2517
                     /label=10 x His
     misc_feature    2524..2568
                     /label=Avi-Tag
     primer_bind     complement(2623..2643)
                     /label=Flexi-Rev
     terminator      2676..2723
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        2952..3056
                     /gene="bla"
                     /label=AmpR promoter
     CDS             3241..3620
                     /label=ISS
                     /note="immunostimulatory sequence from the AmpR gene; 
                     contains unmethylated CpG dinucleotides in the context of 
                     5'-AACGTT-3' (Sato et al., 1996)"
     rep_origin      complement(4076..4664)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     misc_feature    4780..5063
                     /label=cer region
                     /note="ColE1-derived recombination site that helps to 
                     maintain plasmids as monomers"
     terminator      5471..5498
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 ggatctcgat cccgcgaaat taatacgact cactataggg tattttaact ttactaagga
       61 gaattcatca tggcagaaat cggtactggc tttccattcg acccccatta tgtggaagtc
      121 ctgggcgagc gcatgcacta cgtcgatgtt ggtccgcgcg atggcacccc tgtgctgttc
      181 ctgcacggta acccgacctc ctcctacgtg tggcgcaaca tcatcccgca tgttgcaccg
      241 acccatcgct gcattgctcc agacctgatc ggtatgggca aatccgacaa accagacctg
      301 ggttatttct tcgacgacca cgtccgcttc atggatgcct tcatcgaagc cctgggtctg
      361 gaagaggtcg tcctggtcat tcacgactgg ggctccgctc tgggtttcca ctgggccaag
      421 cgcaatccag agcgcgtcaa aggtattgca tttatggagt tcatccgccc tatcccgacc
      481 tgggacgaat ggccagaatt tgcccgcgag accttccagg ccttccgcac caccgacgtc
      541 ggccgcaagc tgatcatcga tcagaacgtt tttatcgagg gtacgctgcc gatgggtgtc
      601 gtccgcccgc tgactgaagt cgagatggac cattaccgcg agccgttcct gaatcctgtt
      661 gaccgcgagc cactgtggcg cttcccaaac gagctgccaa tcgccggtga gccagcgaac
      721 atcgtcgcgc tggtcgaaga atacatggac tggctgcacc agtcccctgt cccgaagctg
      781 ctgttctggg gcaccccagg cgttctgatc ccaccggccg aagccgctcg cctggccaaa
      841 agcctgccta actgcaaggc tgtggacatc ggcccgggtc tgaatctgct gcaagaagac
      901 aacccggacc tgatcggcag cgagatcgcg cgctggctgt cgacgctcga gatttccggc
      961 gagccaacca ctgaggatct gtactttcag agcgataacg cgatcgccgg atccgtaatt
     1021 ggtcttctga ggcctttaaa ggatgtcacc gtgactgcag gggaaacagc caccttcgac
     1081 tgcgagctct cctacgaaga tatcccagtg gaatggtatc tcaaagggaa gaaactagag
     1141 cccagcgata aggtggtccc acgttcagaa ggaaaagttc atacacttac tctgagggat
     1201 gtaaagttag aagatgctgg ggaagtccaa ctaacagcaa aagatttcaa aactcacgcc
     1261 aacctctttg tgaaagaacc cgggtccgta attggtcttc tgaggccttt aaaggatgtc
     1321 accgtgactg caggggaaac agccaccttc gactgcgagc tctcctacga agatatccca
     1381 gtggaatggt atctcaaagg gaagaaacta gagcccagcg ataaggtggt cccacgttca
     1441 gaaggaaaag ttcatacact tactctgagg gatgtaaagt tagaagatgc tggggaagtc
     1501 caactaacag caaaagattt caaaactcac gccaacctct ttgtgaaaga accctccgga
     1561 gcccatgcga cgggtgcggg tccagcgggt cgttatgatc aagccaccga caccatcctc
     1621 accgtgatcg agaacatctt cagcagcatg ggtgatgcgg gcgaaatggt tcgccaagcc
     1681 cgcattctgg cccaagcggt tagcgatctg gtgaatgcga tcaaagccga tgcggaaggt
     1741 gaaagcgatc tggagaacag ccgcaaactg ctcagcgccg ccaaaattct ggcggatgcg
     1801 gttgccaaaa tggttgaagc ggccaaaggt gcggccgcgc atccagatag cgaagaacag
     1861 cagcaacgtc tgcgtgaagc ggccgaaggt ctgcgtatgg cgatcaacgc cgcggcgcag
     1921 aatgccatca aaaaggctag cgtaattggt cttctgaggc ctttaaagga tgtcaccgtg
     1981 actgcagggg aaacagccac cttcgactgc gagctctcct acgaagatat cccagtggaa
     2041 tggtatctca aagggaagaa actagagccc agcgataagg tggtcccacg ttcagaagga
     2101 aaagttcata cacttactct gagggatgta aagttagaag atgctgggga agtccaacta
     2161 acagcaaaag atttcaaaac tcacgccaac ctctttgtga aagaacccgg gtccgtaatt
     2221 ggtcttctga ggcctttaaa ggatgtcacc gtgactgcag gggaaacagc caccttcgac
     2281 tgcgagctct cctacgaaga tatcccagtg gaatggtatc tcaaagggaa gaaactagag
     2341 cccagcgata aggtggtccc acgttcagaa ggaaaagttc atacacttac tctgagggat
     2401 gtaaagttag aagatgctgg ggaagtccaa ctaacagcaa aagatttcaa aactcacgcc
     2461 aacctctttg tgaaagaacc cggaggtcac catcaccatc accatcacca tcaccatggt
     2521 ggcggtctga acgacatctt cgaggctcag aaaatcgaat ggcacgaata aggtaccaga
     2581 tcctctagag tcgacctgca ggcatgcaag ctgatccggc tgctaacaaa gcccgaaagg
     2641 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     2701 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggttcg cttgctgtcc
     2761 ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct
     2821 ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc
     2881 accgtttctg cggactggct ttctacgtaa tggtttctta gacgtcaggt ggcacttttc
     2941 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     3001 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     3061 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     3121 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     3181 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     3241 aacgttttcc aatgatgagc actttcaaag ttctgctatg tggcgcggta ttatcccgta
     3301 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     3361 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     3421 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca actatcggag
     3481 gaccgaagga gctaaccgct tttttgcaca acatgggggg atcatgtaac tcgccttgat
     3541 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     3601 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     3661 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     3721 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     3781 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     3841 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     3901 ctgattaagc attggtaatt cgaaatgacc gaccaagcga cgcccaaccg gtatcagctc
     3961 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4021 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4081 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aagtggcgaa
     4141 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4201 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4261 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4321 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4381 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4441 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4501 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4561 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4621 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatt tcaagaagat cctttgatct
     4681 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4741 gattatcaaa aaggatcttc acctagatcc ttttatagtc tggaaataca ggaacgcacg
     4801 ctggatggcc cttcgctggg atggtgaaac catgaaaaat ggcagcttca gtggattaag
     4861 tgggggtaat gtggcctgta ccctctggtt gcataggtat tcatacggtt aaaatttatc
     4921 aggcgcgatt gcggcagttt ttcgggtggt ttgttgccat ttttacctgt ctgctgccgt
     4981 gatcgcgctg aacgcgtttt agcggtgcgt acaattaagg gattatggta aatccactta
     5041 ctgtctgccc tcgtagccat cgagataaac cgcagtactc cggccacgat gcgtccggcg
     5101 tagaggatcg tgatcttttc agcctgatac agattaaatc agaacgcaga agcggtctga
     5161 taaaacagaa tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact
     5221 cagaagtgaa acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga
     5281 actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc
     5341 tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac
     5401 gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat
     5461 caaattaagc agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttttg
     5521 tttatttttc taaatacatt caaatatgta tccgctcat
//
