LOCUS       sequence-71745-e        4375 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial TD repression YFP reporter: protospacer A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4375)
  AUTHORS   Esvelt KM, Mali P, Braff JL, Moosburner M, Yaung SJ, Church GM
  TITLE     Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.
  JOURNAL   Nat Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681.
  PUBMED    24076762
REFERENCE   2  (bases 1 to 4375)
  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: "Nat 
            Methods. 2013 Sep 29. doi: 10.1038/nmeth.2681."
COMMENT     Sequence Label: SK-YFP-TD-A
FEATURES             Location/Qualifiers
     source          1..4375
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             129..842
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MRKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     complement(159..187)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     782..802
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      904..990
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1525..2475)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTAKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      2523..2745
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3237..3331)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3362..4156)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3473..3492)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4083..4102
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4335..4353
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gatccgataa atatctaaca ccgtgcgtgt tgactatttt acctctggcg gtgataatgg
       61 ttgcatgtac tagccctttt ttcaacaagc ttgagatggt actttaactt taagaaggag
      121 atatacatat gcgtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat
      181 tagatggtga tgttaatggg cacaaatttt ctgtcagtgg agagggtgaa ggtgatgcaa
      241 catacggaaa acttaccctt aaatttattt gcactactgg aaaactacct gttccatggc
      301 caacacttgt cactactttc ggttatggtc taaaatgctt tgctagatac ccagatcata
      361 tgaaacggca tgactttttc aagagtgcca tgcccgaagg ttatgtacag gaaagaacta
      421 tatttttcaa agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata
      481 cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga aacattcttg
      541 gacacaaatt ggaatacaac tataactcac acaatgtata catcatggca gacaaacaaa
      601 agaatggaat caaagttaac ttcaaaatta gacacaacat tgaagatgga agcgttcaac
      661 tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt ttaccagaca
      721 accattacct gtcctatcaa tctgcccttt cgaaagatcc caacgaaaag agagaccaca
      781 tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat gaactataca
      841 aataagctta accgaagcgt ttgatagttg atatcctttg cctgcggccg caactagagg
      901 catcaaataa aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg
      961 tcggtgaacg ctctcctgag taggacaaat ccgccgccct agacctaggg tacgggtttt
     1021 gctgcccgca aacgggctgt tctggtgttg ctagtttgtt atcagaatcg cagatccggc
     1081 ttcaggtttg ccggctgaaa gcgctatttc ttccagaatt gccatgattt tttccccacg
     1141 ggaggcgtca ctggctcccg tgttgtcggc agctttgatt cgataagcag catcgcctgt
     1201 ttcaggctgt ctatgtgtga ctgttgagct gtaacaagtt gtctcaggtg ttcaatttca
     1261 tgttctagtt gctttgtttt actggtttca cctgttctat taggtgttac atgctgttca
     1321 tctgttacat tgtcgatctg ttcatggtga acagctttaa atgcaccaaa aactcgtaaa
     1381 agctctgatg tatctatctt ttttacaccg ttttcatctg tgcatatgga cagttttccc
     1441 tttgatatct aacggtgaac agttgttcta cttttgtttg ttagtcttga tgcttcactg
     1501 atagatacaa gagccataag aacctcagat ccttccgtat ttagccagta tgttctctag
     1561 tgtggttcgt tgtttttgcg tgagccatga gaacgaacca ttgagatcat gcttactttg
     1621 catgtcactc aaaaattttg cctcaaaact ggtgagctga atttttgcag ttaaagcatc
     1681 gtgtagtgtt tttcttagtc cgttacgtag gtaggaatct gatgtaatgg ttgttggtat
     1741 tttgtcacca ttcattttta tctggttgtt ctcaagttcg gttacgagat ccatttgtct
     1801 atctagttca acttggaaaa tcaacgtatc agtcgggcgg cctcgcttat caaccaccaa
     1861 tttcatattg ctgtaagtgt ttaaatcttt acttattggt ttcaaaaccc attggttaag
     1921 ccttttaaac tcatggtagt tattttcaag cattaacatg aacttaaatt catcaaggct
     1981 aatctctata tttgccttgt gagttttctt ttgtgttagt tcttttaata accactcata
     2041 aatcctcata gagtatttgt tttcaaaaga cttaacatgt tccagattat attttatgaa
     2101 tttttttaac tggaaaagat aaggcaatat ctcttcacta aaaactaatt ctaatttttc
     2161 gcttgagaac ttggcatagt ttgtccactg gaaaatctca aagcctttaa ccaaaggatt
     2221 cctgatttcc acagttctcg tcatcagctc tctggttgct ttagctaata caccataagc
     2281 attttcccta ctgatgttca tcatctgagc gtattggtta taagtgaacg ataccgtccg
     2341 ttctttcctt gtagggtttt caatcgtggg gttgagtagt gccacacagc ataaaattag
     2401 cttggtttca tgctccgtta agtcatagcg actaatcgct agttcatttg ctttgaaaac
     2461 aactaattca gacatacatc tcaattggtc taggtgattt taatcactat accaattgag
     2521 atgggctagt caatgataat tactagtcct tttcctttga gttgtgggta tctgtaaatt
     2581 ctgctagacc tttgctggaa aacttgtaaa ttctgctaga ccctctgtaa attccgctag
     2641 acctttgtgt gttttttttg tttatattca agtggttata atttatagaa taaagaaaga
     2701 ataaaaaaag ataaaaagaa tagatcccag ccctgtgtat aactcactac tttagtcagt
     2761 tccgcagtat tacaaaagga tgtcgcaaac gctgtttgct cctctacaaa acagacctta
     2821 aaaccctaaa ggcttaagta gcaccctcgc aagctcgggc aaatcgctga atattccttt
     2881 tgtctccgac catcaggcac ctgagtcgct gtctttttcg tgacattcag ttcgctgcgc
     2941 tcacggctct ggcagtgaat gggggtaaat ggcactacag gcgcctttta tggattcatg
     3001 caaggaaact acccataata caagaaaagc ccgtcacggg cttctcaggg cgttttatgg
     3061 cgggtctgct atgtggtgct atctgacttt ttgctgttca gcagttcctg ccctctgatt
     3121 ttccagtctg accacttcgg attatcccgt gacaggtcat tcagactggc taatgcaccc
     3181 agtaaggcag cggtatcatc aacaggctta cccgtcttac tgtccctagt gcttggattc
     3241 tcaccaataa aaaacgcccg gcggcaaccg agcgttctga acaaatccag atggagttct
     3301 gaggtcatta ctggatctat caacaggagt ccaagcgagc tctcgaaccc cagagtcccg
     3361 ctcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga
     3421 taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac
     3481 gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga
     3541 atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca
     3601 cgacgagatc ctcgccgtcg ggcatgcgcg ccttgagcct ggcgaacagt tcggctggcg
     3661 cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag
     3721 tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa
     3781 gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt
     3841 gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt
     3901 cagtgacaac gtcgagcaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc
     3961 gcgctgcctc gtcctgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa
     4021 ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt
     4081 gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc
     4141 catcttgttc aatcatgcga aacgatcctc atcctgtctc ttgatcagat cttgatcccc
     4201 tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag ggcttcccaa
     4261 ccttaccaga gggcgcccca gctggcaatt ccgacgtcta agaaaccatt attatcatga
     4321 cattaaccta taaaaatagg cgtatcacga ggccctttcg tcttcacctc gaggg
//
