LOCUS       62056_19180        6710 bp DNA     circular SYN 15-OCT-2021
DEFINITION  Cloning vector pSAG1-mNeonGreen_TUB1-dTomato, complete sequence.
ACCESSION   MZ090944
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6710)
  AUTHORS   Markus BM, Boydston EA, Lourido S.
  TITLE     CRISPR-Mediated Transcriptional Repression in Toxoplasma gondii
  JOURNAL   mSphere, e0047421 (2021) In press
  PUBMED    34643425
REFERENCE   2  (bases 1 to 6710)
  AUTHORS   Markus BM.
  TITLE     Direct Submission
  JOURNAL   Submitted (02-MAY-2021) Whitehead Institute, 455 Main Street, 
            Cambridge, MA 02142, United States
REFERENCE   3  (bases 1 to 6710)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "mSphere, 
            e0047421 (2021) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (02-MAY-2021) Whitehead Institute, 455 Main Street, Cambridge, MA 
            02142, United States"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..6710
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    672..1428
                     /label=TgSAG1 upstream region
                     /note="TgSAG1 upstream region"
     CDS             1435..2142
                     /label=mNeonGreen
                     /note="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
     misc_feature    2143..2172
                     /label=ty1 tag
                     /note="ty1 tag"
     gene            2184..2496
                     /gene="TgSAG1"
                     /label=TgSAG1
     3'UTR           2184..2496
                     /gene="TgSAG1"
     misc_feature    2540..3037
                     /label=TgTUB1 upstream region
                     /note="TgTUB1 upstream region"
     CDS             3044..3745
                     /label=dTomato
                     /note="dimeric variant of DsRed fluorescent protein (Shaner
                     et al., 2004)"
     gene            3755..4496
                     /gene="TgDHFR-TS"
                     /label=TgDHFR-TS
     3'UTR           3755..4496
                     /gene="TgDHFR-TS"
     promoter        complement(4522..4540)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(4561..4577)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4585..4601)
                     /label=lac operator
                     /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(4609..4639)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4654..4675)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4963..5551)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(5725..6582)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(6583..6687)
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccgggcc
      661 ccccctcgag gaagctttta catccgttgc cttttccacg gtccgtgatt tcatgtgcgt
      721 gcagcttcaa agactggtcg ttgcgactaa taagactgca gtgacaggtc gaatggtggg
      781 caccttgctg atgactatct actgcaaagt ctgagacaac gaacgaaact tcccacacga
      841 ggcatttgaa actgacggtg tctaggtaat atgcactgca agacacggta ctggggcctc
      901 gctgaattag gggccgatct cgttgcccta tcagtgctca cagtgccgca acgtaacacc
      961 agggcaggtt cttgacagtg gcaacaatgt gcgacgggcg tgtgaacgtt tcgtagtcat
     1021 agcgctagca cgtacctagc cacatggtcg tgaggagctt taccatgcgt ctagaaggtg
     1081 gatgcgggac acgccttcct ggcctttggc tcccgagacg cgtgttctaa ccacaaacct
     1141 tgagacgcgt gttccaacca cgcaccctga cacgcgtgtt ccaaccacgc accctgagac
     1201 gcgtgttcta accacgcacc ctgagacgcg tgttctaacc acgcaccctg agacgcgtgt
     1261 tctgccgcac aatgtgcacc tgtaggaagc tgtagtcact gctgattctc actgttctcg
     1321 gcaagggccg acgaccggag tacagttttt gtgggcagag ccgttgtgca gctttccgtt
     1381 cttctcggtt gtgtcacatg tgtcattgtc gtgtaaacac acggttgtga attcatggtg
     1441 agcaagggcg aggaggataa catggcctct ctcccagcga cacatgagtt acacatcttt
     1501 ggctccatca acggtgtgga ctttgacatg gtgggtcagg gcaccggcaa tccaaatgat
     1561 ggttatgagg agttaaacct gaagtccacc aagggtgacc tccagttctc cccctggatt
     1621 ctggtccctc atatcgggta tggcttccat cagtacctgc cctaccctga cgggatgtcg
     1681 cctttccagg ccgccatggt agatggctcc ggctaccaag tccatcgcac aatgcagttt
     1741 gaagatggtg cctcccttac tgttaactac cgctacacct acgagggaag ccacatcaaa
     1801 ggagaggccc aggtgaaggg gactggtttc cctgctgacg gtcctgtgat gaccaactcg
     1861 ctgaccgctg cggactggtg caggtcgaag aagacttacc ccaacgacaa aaccatcatc
     1921 agtaccttta agtggagtta caccactgga aatggcaagc gctaccggag cactgcgcgg
     1981 accacctaca cctttgccaa gccaatggcg gctaactatc tgaagaacca gccgatgtac
     2041 gtgttccgta agacggagct caagcactcc aagaccgagc tcaacttcaa ggagtggcaa
     2101 aaggccttta ccgatgtgat gggcatggac gagctgtaca aggaggtcca cacgaaccag
     2161 gacccgctcg attaagttaa ttaatcaccg ttgtgctcac ttctcaaatc gacaaaggaa
     2221 acacacttcg tgcagcatgt gccccattat aaagaaactg agttgttccg ntgtggcttg
     2281 caggtgtcac atccacaaaa accggccgac tctaaatagg agtgtttcgc agcaagcagc
     2341 gaaagtttat gactgggtcc gaatctctga acggatgtgt ggcggacctg gctgatgttg
     2401 atcgccgtcg acacacgcgc cacatgggtc aatacacaag acagctatca gttgttttag
     2461 tcgaaccggt taacacaatt cttgcccccc cgagggacta gtctggacgg tcgacggtat
     2521 cgataagctt gtagcatgcc atgcatgtcc cgcgttcgtg aaattctctg catcagcgga
     2581 gtgatcagga atcatcgtct cagcgggatg acgttgcgga gcaggccggc tcgcggtggg
     2641 cagtcagatg ccgaaggcgt aactcaggac ggcttgcgct catcgcagaa caggggtggt
     2701 gcctgcattg ggtgcggttg gtgatcctgg ttggaccggt ggagatgcgc gcgcacgaag
     2761 gggatgtgtc agaaacattt tgtttgttct ctgtgaactt ttagatgtgt taaaggcggc
     2821 gaatattanc agagagtcct ccttgttgga ttctctcttg aatttcgccc tttccttctc
     2881 tttgcgagtc tcgtagagaa caagcactcg ttcgccgtcc ctgacgacgc aacccgcgca
     2941 gaagacatcc accaaacggt gttacacaat caccttgtgt gaagttcttg cggaaaacta
     3001 ctcgttggca ttttttcttg aattcccttt ttcgacacct aggatggtga gcaagggcga
     3061 ggaggtcatc aaagagttca tgcgcttcaa ggtgcgcatg gagggctcca tgaacggcca
     3121 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     3181 gctgaaggtg accaagggcg gccccctgcc cttcgcctgg gacatcctgt ccccccagtt
     3241 catgtacggc tccaaggcgt acgtgaagca ccccgccgac atccccgatt acaagaagct
     3301 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggtctggt
     3361 gaccgtgacc caggactcct ccctgcagga cggcacgctg atctacaagg tgaagatgcg
     3421 cggcaccaac ttcccccccg acggccccgt aatgcagaag aagaccatgg gctgggaggc
     3481 ctccaccgag cgcctgtacc cccgcgacgg cgtgctgaag ggcgagatcc accaggccct
     3541 gaagctgaag gacggcggcc actacctggt ggagttcaag acgatctaca tggccaagaa
     3601 gcccgtgcaa ctgcccggct actactacgt ggacaccaag ctggacatca cctcccacaa
     3661 cgaggactac accatcgtgg aacagtacga gcgctccgag ggccgccacc acctgttcct
     3721 gtacggcatg gacgagctgt acaagtaaca attgacagaa gctgcccgtc tctcgttttc
     3781 ctctcttttc ggagggatca gggagagtgc ctcgggtcgg agagagctga cgagggggtg
     3841 ccagagaccc ctgtgtcctt tatcgaagaa aagggatgac tcttcatgtg gcatttcaca
     3901 cagtctcacc tcgccttgtt ttctttttgt caatcagaac gaaagcgagt tgcgggtgac
     3961 gcagatgtgc gtgtatccac tcgngaatgc gttatcgttc tgtatgccgc tagagtgctg
     4021 gactgttgct gtctgcccac gacagcagac aactttcctt ctatgcactt gcaggatggt
     4081 gcagcgcaaa cgacggagag aaaggagcac cctctcagtt tccctacgat gtgctgtcag
     4141 tttcgactct tcaccgcgaa cgattggcga tacgtctctg ttgacttgtt aggctccgac
     4201 cacgaagctc ccttaactag ataagccgcg acacctaagt gtacaccatt tgcagatcga
     4261 taatctgcga ccgctgaatc cgtccagatc agtaaaaccg caccacctaa gtgtaaacct
     4321 tgtttaggtc gataaaatgc taccaacccc cacccacaat cgagccttga gcgtttctgc
     4381 gcacgcgttg gcctacgtga cttgctgatg cctgcctctg gccattcatg ccagtcagtg
     4441 cgcataaaaa tgtggacaca gtcggttgac aagtgttctg gcaggctaca tgacacgcgg
     4501 ccgcagctcc agcttttgtt ccctttagtg agggttaatt tcgagcttgg cgtaatcatg
     4561 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4621 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     4681 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4741 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4801 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4861 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4921 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     4981 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5041 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5101 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5161 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5221 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5281 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5341 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5401 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5461 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5521 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5581 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5641 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5701 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5761 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5821 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5881 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     5941 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6001 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6061 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6121 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6181 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6241 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6301 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6361 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6421 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6481 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6541 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6601 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6661 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
//
