LOCUS       sequence-431542-        3224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fragmid fragment: (Cas protein) binds to TRE in absence of 
            doxycycline.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3224)
  AUTHORS   McGee AV, Liu YV, Griffith AL, Szegletes ZM, Wen B, Kraus C, Miller 
            NW, Steger RJ, Escude Velasco B, Bosch JA, Zirin JD, Viswanatha R, 
            Sontheimer EJ, Goodale A, Greene MA, Green TM, Doench JG
  TITLE     Modular vector assembly enables rapid assessment of emerging CRISPR 
            technologies.
  JOURNAL   Cell Genom. 2024 Mar 13;4(3):100519. doi: 
            10.1016/j.xgen.2024.100519.
  PUBMED    38484704
REFERENCE   2  (bases 1 to 3224)
  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"; journalName: "Cell 
            Genom."; date: "2024-03-13"; volume: "4(3):100519. doi"; pages: " 
            10.1016/j.xgen.2024.100519"
COMMENT     Sequence Label: AA440
FEATURES             Location/Qualifiers
     source          1..3224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             448..1062
                     /codon_start=1
                     /gene="tetR from transposon Tn10"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="RLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKN
                     KRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPT
                     EKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDS
                     MPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     primer_bind     complement(515..534)
                     /label=Tet-R
                     /note="Tetracycline resistance gene, reverse primer"
     primer_bind     complement(1134..1150)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1134..1150)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1147..1169)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1158..1174
                     /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(1182..1188,1189..1206,1207..1212))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1227..1248
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1365..1382)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1536..2124)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1616..1635)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2302..3111)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     3025..3044
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3103..3122
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagagaatt cgagctcggt acctcgcgaa
      421 tacatctaga tgaagactgg cagcgctaga ttagataaaa gtaaagtgat taacagcgca
      481 ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa cccgtaaact cgcccagaag
      541 ctaggtgtag agcagcctac attgtattgg catgtaaaaa ataagcgggc tttgctcgac
      601 gccttagcca ttgagatgtt agataggcac catactcact tttgcccttt agaaggggaa
      661 agctggcaag attttttacg taataacgct aaaagtttta gatgtgcttt actaagtcat
      721 cgcgatggag caaaagtaca tttaggtaca cggcctacag aaaaacagta tgaaactctc
      781 gaaaatcaat tagccttttt atgccaacaa ggtttttcac tagagaatgc attatatgca
      841 ctcagcgctg tggggcattt tactttaggt tgcgtattgg aagatcaaga gcatcaagtc
      901 gctaaagaag aaagggaaac acctactact gatagtatgc cgccattatt acgacaagct
      961 atcgaattat ttgatcacca aggtgcagag ccagccttct tattcggcct tgaattgatc
     1021 atttgcggat tagaaaaaca acttaaatgt gaaagtggtt ccgggtccaa gtcttcatcg
     1081 gatcccgggc ccgtcgactg cagaggcctg catgcaagct tggtgtaatc atggtcatag
     1141 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1201 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1261 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1321 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1381 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     1441 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     1501 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     1561 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     1621 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     1681 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     1741 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     1801 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     1861 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     1921 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     1981 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2041 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2101 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2161 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2221 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa gcccaatctg aataatgtta
     2281 caaccaatta accaattctg attagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     2341 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     2401 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     2461 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     2521 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     2581 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     2641 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     2701 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     2761 attttcacct gaatcaggat attcttctaa tacctggaat gctgtttttc cggggatcgc
     2821 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     2881 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     2941 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca agcgatagat
     3001 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     3061 catgttggaa tttaatcgcg gcctcgacgt ttcccgttga atatggctca taacacccct
     3121 tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg
     3181 tgcaatgtaa catcagagat tttgagacac gggccagagc tgca
//
