LOCUS       sequence-71740-e        4381 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial TD repression YFP reporter: protospacer B.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4381)
  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 4381)
  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-B
FEATURES             Location/Qualifiers
     source          1..4381
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             141..848
                     /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="KGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFI
                     CTTGKLPVPWPTLVTTFGYGLKCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDGNY
                     KTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNF
                     KIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFV
                     TAAGITHGMDELYK"
     primer_bind     complement(165..193)
                     /label=GFP-R
                     /note="GFP, reverse primer. Does NOT anneal to EGFP"
     primer_bind     788..808
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     terminator      910..996
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1531..2481)
                     /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      2529..2751
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      complement(3243..3337)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(3368..4162)
                     /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(3479..3498)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     4089..4108
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4341..4359
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 gatccgataa atatctaaca ccgtgcgtgt tgactatttt acctctggcg gtgataatgg
       61 ttgcatgtac tagaattctt taactttaag aaggagatat acatatgaag ttttcatggc
      121 gaatactttt aaagtctcgt aaaggagaag aacttttcac tggagttgtc ccaattcttg
      181 ttgaattaga tggtgatgtt aatgggcaca aattttctgt cagtggagag ggtgaaggtg
      241 atgcaacata cggaaaactt acccttaaat ttatttgcac tactggaaaa ctacctgttc
      301 catggccaac acttgtcact actttcggtt atggtctaaa atgctttgct agatacccag
      361 atcatatgaa acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaaa
      421 gaactatatt tttcaaagat gacgggaact acaagacacg tgctgaagtc aagtttgaag
      481 gtgataccct tgttaataga atcgagttaa aaggtattga ttttaaagaa gatggaaaca
      541 ttcttggaca caaattggaa tacaactata actcacacaa tgtatacatc atggcagaca
      601 aacaaaagaa tggaatcaaa gttaacttca aaattagaca caacattgaa gatggaagcg
      661 ttcaactagc agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac
      721 cagacaacca ttacctgtcc tatcaatctg ccctttcgaa agatcccaac gaaaagagag
      781 accacatggt ccttcttgag tttgtaacag ctgctgggat tacacatggc atggatgaac
      841 tatacaaata agcttaaccg aagcgtttga tagttgatat cctttgcctg cggccgcaac
      901 tagaggcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt
      961 tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgccctagac ctagggtacg
     1021 ggttttgctg cccgcaaacg ggctgttctg gtgttgctag tttgttatca gaatcgcaga
     1081 tccggcttca ggtttgccgg ctgaaagcgc tatttcttcc agaattgcca tgattttttc
     1141 cccacgggag gcgtcactgg ctcccgtgtt gtcggcagct ttgattcgat aagcagcatc
     1201 gcctgtttca ggctgtctat gtgtgactgt tgagctgtaa caagttgtct caggtgttca
     1261 atttcatgtt ctagttgctt tgttttactg gtttcacctg ttctattagg tgttacatgc
     1321 tgttcatctg ttacattgtc gatctgttca tggtgaacag ctttaaatgc accaaaaact
     1381 cgtaaaagct ctgatgtatc tatctttttt acaccgtttt catctgtgca tatggacagt
     1441 tttccctttg atatctaacg gtgaacagtt gttctacttt tgtttgttag tcttgatgct
     1501 tcactgatag atacaagagc cataagaacc tcagatcctt ccgtatttag ccagtatgtt
     1561 ctctagtgtg gttcgttgtt tttgcgtgag ccatgagaac gaaccattga gatcatgctt
     1621 actttgcatg tcactcaaaa attttgcctc aaaactggtg agctgaattt ttgcagttaa
     1681 agcatcgtgt agtgtttttc ttagtccgtt acgtaggtag gaatctgatg taatggttgt
     1741 tggtattttg tcaccattca tttttatctg gttgttctca agttcggtta cgagatccat
     1801 ttgtctatct agttcaactt ggaaaatcaa cgtatcagtc gggcggcctc gcttatcaac
     1861 caccaatttc atattgctgt aagtgtttaa atctttactt attggtttca aaacccattg
     1921 gttaagcctt ttaaactcat ggtagttatt ttcaagcatt aacatgaact taaattcatc
     1981 aaggctaatc tctatatttg ccttgtgagt tttcttttgt gttagttctt ttaataacca
     2041 ctcataaatc ctcatagagt atttgttttc aaaagactta acatgttcca gattatattt
     2101 tatgaatttt tttaactgga aaagataagg caatatctct tcactaaaaa ctaattctaa
     2161 tttttcgctt gagaacttgg catagtttgt ccactggaaa atctcaaagc ctttaaccaa
     2221 aggattcctg atttccacag ttctcgtcat cagctctctg gttgctttag ctaatacacc
     2281 ataagcattt tccctactga tgttcatcat ctgagcgtat tggttataag tgaacgatac
     2341 cgtccgttct ttccttgtag ggttttcaat cgtggggttg agtagtgcca cacagcataa
     2401 aattagcttg gtttcatgct ccgttaagtc atagcgacta atcgctagtt catttgcttt
     2461 gaaaacaact aattcagaca tacatctcaa ttggtctagg tgattttaat cactatacca
     2521 attgagatgg gctagtcaat gataattact agtccttttc ctttgagttg tgggtatctg
     2581 taaattctgc tagacctttg ctggaaaact tgtaaattct gctagaccct ctgtaaattc
     2641 cgctagacct ttgtgtgttt tttttgttta tattcaagtg gttataattt atagaataaa
     2701 gaaagaataa aaaaagataa aaagaataga tcccagccct gtgtataact cactacttta
     2761 gtcagttccg cagtattaca aaaggatgtc gcaaacgctg tttgctcctc tacaaaacag
     2821 accttaaaac cctaaaggct taagtagcac cctcgcaagc tcgggcaaat cgctgaatat
     2881 tccttttgtc tccgaccatc aggcacctga gtcgctgtct ttttcgtgac attcagttcg
     2941 ctgcgctcac ggctctggca gtgaatgggg gtaaatggca ctacaggcgc cttttatgga
     3001 ttcatgcaag gaaactaccc ataatacaag aaaagcccgt cacgggcttc tcagggcgtt
     3061 ttatggcggg tctgctatgt ggtgctatct gactttttgc tgttcagcag ttcctgccct
     3121 ctgattttcc agtctgacca cttcggatta tcccgtgaca ggtcattcag actggctaat
     3181 gcacccagta aggcagcggt atcatcaaca ggcttacccg tcttactgtc cctagtgctt
     3241 ggattctcac caataaaaaa cgcccggcgg caaccgagcg ttctgaacaa atccagatgg
     3301 agttctgagg tcattactgg atctatcaac aggagtccaa gcgagctctc gaaccccaga
     3361 gtcccgctca gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag
     3421 cggcgatacc gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa
     3481 tatcacgggt agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt
     3541 cgatgaatcc agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat
     3601 gggtcacgac gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg
     3661 ctggcgcgag cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca
     3721 tccgagtacg tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg
     3781 gatcaagcgt atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag
     3841 caaggtgaga tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc
     3901 ccgcttcagt gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg
     3961 atagccgcgc tgcctcgtcc tgcagttcat tcagggcacc ggacaggtcg gtcttgacaa
     4021 aaagaaccgg gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg
     4081 tctgttgtgc ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt
     4141 gcaatccatc ttgttcaatc atgcgaaacg atcctcatcc tgtctcttga tcagatcttg
     4201 atcccctgcg ccatcagatc cttggcggca agaaagccat ccagtttact ttgcagggct
     4261 tcccaacctt accagagggc gccccagctg gcaattccga cgtctaagaa accattatta
     4321 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt cacctcgagg
     4381 g
//
