LOCUS       Exported                2322 bp ds-DNA     circular SYN 21-FEB-2022
DEFINITION  .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2322)
  AUTHORS   Carolyn Huang
  TITLE     Direct Submission
  JOURNAL   Exported Tuesday, Feb 22, 2022 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..2322
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     terminator      5..22
                     /label=L3S2P55 Terminator (Voigt)
                     /note="A strong synthetic promoter from Voigt's Nature 
                     Methods paper
                     
                     Also used by Alex Nielsen as a dSpyCas9 sgRNA terminator in
                     his CRISPRi logic gates paper
                     "
                     /note="color: #ffffff; direction: RIGHT"
     protein_bind    23..26
                     /label=BsaI
                     /note="color: #00ff2c"
     misc_feature    27..51
                     /label=ConE scar
                     /note="color: #fffd6c"
     protein_bind    27..30
                     /label=BsmBI
                     /note="color: #00ff15"
     terminator      complement(71..127)
                     /label=ECK120033737 Terminator (Voigt)
                     /note="rho-independent terminator of thrLABC operon"
                     /note="color: #ffffff; direction: LEFT"
     misc_feature    complement(128..236)
                     /label=CamR Terminator
                     /note="color: #66ccff; direction: LEFT"
     CDS             complement(237..896)
                     /label=CamR
                     /note="color: #0000ff; direction: LEFT"
     promoter        complement(897..1001)
                     /label=CamR Promoter
                     /note="color: #66ccff; direction: LEFT"
     rep_origin      complement(1269..1813)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
                     /note="color: #ffff00"
     terminator      1914..2003
                     /label=ECK120029600 Terminator (Voigt)
                     /note="rho-independent terminator of spheroplast protein Y 
                     transcript"
                     /note="color: #ffffff; direction: RIGHT"
     misc_feature    2023..2047
                     /label=ConS scar
                     /note="color: #fffd6c"
     protein_bind    2023..2026
                     /label=BsmBI
                     /note="color: #00ff15"
     misc_feature    2048..2051
                     /label=Pt2/2a BsaI Junction Scar
                     /note="color: #800080"
     promoter        2052..2149
                     /label=proD Promoter (strong constitutive)
                     /note="from ""Design, construction and characterization of 
                     a set of insulated bacterial promoters""
                     
                     The paper says this is synthetic. In other words, I assumed
                     this was from a ProD gene, but it does not appear to be the
                     case; specifically, the proline pathway in E. coli has 
                     genes proABC but not proD...
                     "
                     /note="This forward directional feature has 6 segments:
                        1: 2052 .. 2113 / #339966
                        2: 2114 .. 2119 / #00ff00 / -35
                        3: 2120 .. 2136 / #339966
                        4: 2137 .. 2141 / #00ff00 / -10
                        5: 2142 .. 2148 / #339966
                        6: 2149 .. 2149 / #00ff00"
     misc_RNA        2152..2283
                     /label=transcript
                     /note="color: #a6acb3; direction: RIGHT"
     misc_feature    2152..2226
                     /label=Cas12c_tracrRNA
                     /note="color: #ffcc99; direction: RIGHT"
     misc_feature    2227..2230
                     /label=Tetraloop
                     /note="color: #0000ff"
     misc_feature    2231..2255
                     /label=Repeat
                     /note="Cas12c.1"
                     /note="color: #339966"
     misc_feature    complement(2256..2272)
                     /label=Strutt SauCas9 noPAM GFP2
                     /note="This reverse directional feature has 2 segments:
                        1: 2256 .. 2259 / #008000
                        2: 2260 .. 2272 / #339966"
     misc_feature    2256..2272
                     /label=spacer 1 targeting Qi sfGFP
                     /label=sfGFP
                     /note="ApEinfo_revcolor=green
                     ApEinfo_fwdcolor=#00f700
                     ApEinfo_graphicformat=arrow_data {{0 1 2 0 0 -1} {} 0} 
                     width 5 offset 0"
                     /note="color: #00f700"
     misc_feature    complement(2256..2261)
                     /label=SauCas9 PAM (less stringent)
                     /note="color: #3366ff; direction: LEFT"
     misc_feature    complement(2256..2260)
                     /label=SauCas9 PAM (less stringent)
                     /note="color: #3366ff; direction: LEFT"
     misc_feature    complement(2257..2272)
                     /label=NGG PAM SpyCas9 GFP1 (Best Qi)
                     /note="This reverse directional feature has 2 segments:
                        1: 2257 .. 2259 / #ff0000 / PAM
                        2: 2260 .. 2272 / #00ff00 / Spacer"
     misc_feature    complement(2261..2266)
                     /label=SauCas9 PAM
                     /note="color: #0000ff; direction: LEFT"
     misc_feature    2268..2272
                     /label=SauCas9 PAM
                     /note="color: #0000ff; direction: RIGHT"
     misc_feature    2273..2283
                     /label=11nt Repeat
                     /note="color: #339966"
     terminator      join(2284..2322,1..22)
                     /label=L3S2P21 Terminator (Voigt)
                     /note="Strongest synthetic promoter from Voigt's Nature 
                     Methods paper
                     
                     Also used by Alex Nielsen as a dSpyCas9 sgRNA terminator in
                     his CRISPRi logic gates paper
                     "
                     /note="color: #ffffff; direction: RIGHT"
ORIGIN
        1 gggccttttt tcgttttggt ccgctgagca gttacagaga tgttacgaac cactagtgca
       61 ctgcagtaca cctttggtcg aaaaaaaaag cccgcactgt caggtgcggg cttttttctg
      121 tgtttccacc aataaaaaac gcccggcggc aaccgagcgt tctgaacaaa tccagatgga
      181 gttctgaggt cattactgga tctatcaaca ggagtccaag cgagctcgat atcaaattac
      241 gccccgccct gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg
      301 aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct
      361 tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg
      421 tttaaatcaa aactggtgaa actcacccag ggattggctg aaacgaaaaa catattctca
      481 ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat
      541 atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca
      601 gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg
      661 tctttcattg ccatacgaaa ttccggatga gcattcatca ggcgggcaag aatgtgaata
      721 aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc
      781 agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct
      841 ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta
      901 gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt
      961 tcattatggt gaaagttgga acctcttacg tgcccgatca agcgctagcg gagtgtatac
     1021 tggcttacta tgttggcact gatgagggtg tcagtgaagt gcttcatgtg gcaggagaaa
     1081 aaaggctgca ccggtgcgtc agcagaatat gtgatacagg atatattccg cttcctcgct
     1141 cactgactcg ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg
     1201 cggagatttc ctggaagatg ccaggaagat acttaacagg gaagtgagag ggccgcggca
     1261 aagccgtttt tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat
     1321 cagtggtggc gaaacccgac aggactataa agataccagg cgtttcccct ggcggctccc
     1381 tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc
     1441 gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac
     1501 tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt
     1561 gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt
     1621 agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg
     1681 tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt
     1741 cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc
     1801 aaaacgatct caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca
     1861 atttatctct tcaaatgtag cacctgaagt cagccccata cgatataagt tgtttcagcc
     1921 aaaaaactta agaccgccgg tcttgtccac taccttgcag taatgcggtg gacaggatcg
     1981 gcggttttct tttctcttct caaccctgaa ttcgcatcta gactgaactg gccgataatt
     2041 gcagacgaac gcacagctaa caccacgtcg tccctatctg ctgccctagg tctatgagtg
     2101 gttgctggat aactttacgg gcatgcataa ggctcgtata atatattcag gataccaccc
     2161 gtgcatttct ggatcaatga tccgtacctc aatgtccggg cgcgcagcta gagcgacctg
     2221 aaatctgaaa agcaggattc aggttgggtt tgaggtccca attcttgttg aaagcaggat
     2281 tcactcggta ccaaattcca gaaaagaggc ctcccgaaag gg
//
