LOCUS       sequence-461260-        5875 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  encodes a YFP-tubulin fusion contruct under the control of a dox 
            inducible promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5875)
  AUTHORS   Pelosse M, Marcia M
  TITLE     biGMamAct: efficient CRISPR/Cas9-mediated docking of large 
            functional DNA cargoes at the ACTB locus
  JOURNAL   Synthetic Biology 2025.02.13
REFERENCE   2  (bases 1 to 5875)
  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: "Synthetic 
            Biology 2025.02.13"
COMMENT     Sequence Label: psLIB TRE EYFP-tubulin
FEATURES             Location/Qualifiers
     source          1..5875
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      123..711
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     612..631
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     865..882
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  1016..1240
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(1307..1840)
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     promoter        complement(join(2029..2034,2035..2051,2052..2057))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    complement(2453..2486)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2566..2584
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2603..2621
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2640..2658
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2677..2695
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2714..2732
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2751..2769
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2788..2806
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2825..2843
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2865..2889
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      3047..3056
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3053..3055,3056..3058,3059..3769)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3098..3119)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3359..3378)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3706..3727
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    5344..5478
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5381..5400)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5435..5454
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  5588..5753
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
ORIGIN
        1 ttattaatgt ttgtaattga ctgaatatca acgcttattt gcagcctgaa tggcgaatgg
       61 gacgcgcccg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
      121 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
      181 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
      241 ttcagcagag cgcagatacc aaatactatt cttctagtgt agccgtagtt aggccaccac
      301 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
      361 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
      421 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
      481 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
      541 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      601 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      661 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      721 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      781 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      841 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
      901 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcataga ccagccgcgt
      961 aacctggcaa aatcggttac ggttgagtaa taaatggatg ccctgcgtaa gcgggtgtgg
     1021 gcggacaata aagtcttaaa ctgaacaaaa tagatctaaa ctatgacaat aaagtcttaa
     1081 actagacaga atagttgtaa actgaaatca gtccagttat gctgtgaaaa agcatactgg
     1141 acttttgtta tggctaaagc aaactcttca ttttctgaag tgcaaattgc ccgtcgtatt
     1201 aaagaggggc gtggccaagg gcatggtaaa gactatattc gcggcgttgt gacaatttac
     1261 cgaacaactc cgcggccggg aagccgatct cggcttgaac gaattgttag gtggcggtac
     1321 ttgggtcgat atcaaagtgc atcacttctt cccgtatgcc caactttgta tagagagcca
     1381 ctgcgggatc gtcaccgtaa tctgcttgca cgtagatcac ataagcacca agcgcgttgg
     1441 cctcatgctt gaggagattg atgagcgcgg tggcaatgcc ctgcctccgg tgctcgccgg
     1501 agactgcgag atcatagata tagatctcac tacgcggctg ctcaaacttg ggcagaacgt
     1561 aagccgcgag agcgccaaca accgcttctt ggtcgaaggc agcaagcgcg atgaatgtct
     1621 tactacggag caagttcccg aggtaatcgg agtccggctg atgttgggag taggtggcta
     1681 cgtctccgaa ctcacgaccg aaaagatcaa gagcagcccg catggatttg acttggtcag
     1741 ggccgagcct acatgtgcga atgatgccca tacttgagcc acctaacttt gttttagggc
     1801 gactgccctg ctgcgtaaca tcgttgctgc tgcgtaacat cgttgctgct ccataacatc
     1861 aaacatcgac ccacggcgta acgcgcttgc tgcttggatg cccgaggcat agactgtaca
     1921 aaaaaacagt cataacaagc catgaaaacc gccactgcgc cgttaccacc gctgcgttcg
     1981 gtcaaggttc tggaccagtt gcgtgagcgc atacgctact tgcattacag tttacgaacc
     2041 gaacaggctt atgtcaactg ggtttgtgcc ttcatccgtt tccacggtgt gcgtcacccg
     2101 gcaaccttgg gcagcagcga agtcgaggca tttctgtcct ggctggcgaa cgagcgcaag
     2161 gtttcggtct ccacgcatcg tcaggcattg gcggccttgc tgttcttcta cggcaaggtg
     2221 ctgtgcacgg atctgccctt gcttcaggag atcggtagac ctcggccgtc gcggcgcttg
     2281 ccggtggtgc tgaccccgga tgaagtggtt cgcatcctcg gttttctgga aggcgagcat
     2341 cgtttgttcg cccaggactc tagctatagt tctagtggtt ggctacagct ttgtttgtac
     2401 tatcaacagg ttgaactgct gatcaacaga tcctctacgc ggccgcggta ccataacttc
     2461 gtatagcata cattatacga agttatctgg ttttactagt atcgattcgc gacctactcc
     2521 ggaatattaa tagatcactc gagggatcca agctggccgc acgtctccct atcagtgata
     2581 gagaagtcga cacgtctcga gctccctatc agtgatagag aaggtacgtc tagaacgtct
     2641 ccctatcagt gatagagaag tcgacacgtc tcgagctccc tatcagtgat agagaaggta
     2701 cgtctagaac gtctccctat cagtgataga gaagtcgaca cgtctcgagc tccctatcag
     2761 tgatagagaa ggtacgtcta gaacgtctcc ctatcagtga tagagaagtc gacacgtctc
     2821 gagctcccta tcagtgatag agaaggtacc ccctatataa gcagagctcg tttagtgaac
     2881 cgtcagatcg cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac
     2941 cgatccagcc tccgcggtgg cggccatcac aagtcatacc gtcccaccat cgggcgcgga
     3001 tcccggtccg aagcgcgcgg aattcaaagg gctagcgcta ccggtcgcca ccatggtgag
     3061 caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt
     3121 aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct
     3181 gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac
     3241 caccttcggc tacggcctgc agtgcttcgc ccgctacccc gaccacatga agcagcacga
     3301 cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga
     3361 cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg
     3421 catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga
     3481 gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa
     3541 ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta
     3601 ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag
     3661 ctaccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga
     3721 gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt ccggactcag
     3781 atctcgagtg cgtgagtgca tctccatcca cgttggccag gctggtgtcc agattggcaa
     3841 tgcctgctgg gagctctact gcctggaaca cggcatccag cccgatggcc agatgccaag
     3901 tgacaagacc attgggggag gagatgactc cttcaacacc ttcttcagtg agacgggcgc
     3961 tggcaagcac gtgccccggg ctgtgtttgt agacttggaa cccacagtca ttgatgaagt
     4021 tcgcactggc acctaccgcc agctcttcca ccctgagcag ctcatcacag gcaaggaaga
     4081 tgctgccaat aactatgccc gagggcacta caccattggc aaggagatca ttgaccttgt
     4141 gttggaccga attcgcaagc tggctgacca gtgcaccggt cttcagggct tcttggtttt
     4201 ccacagcttt ggtgggggaa ctggttctgg gttcacctcc ctgctcatgg aacgtctctc
     4261 agttgattat ggcaagaagt ccaagctgga gttctccatt tacccagcac cccaggtttc
     4321 cacagctgta gttgagccct acaactccat cctcaccacc cacaccaccc tggagcactc
     4381 tgattgtgcc ttcatggtag acaatgaggc catctatgac atctgtcgta gaaacctcga
     4441 tatcgagcgc ccaacctaca ctaaccttaa ccgccttatt agccagattg tgtcctccat
     4501 cactgcttcc ctgagatttg atggagccct gaatgttgac ctgacagaat tccagaccaa
     4561 cctggtgccc tacccccgca tccacttccc tctggccaca tatgcccctg tcatctctgc
     4621 tgagaaagcc taccatgaac agctttctgt agcagagatc accaatgctt gctttgagcc
     4681 agccaaccag atggtgaaat gtgaccctcg ccatggtaaa tacatggctt gctgcctgtt
     4741 gtaccgtggt gacgtggttc ccaaagatgt caatgctgcc attgccacca tcaaaaccaa
     4801 gcgcagcatc cagtttgtgg attggtgccc cactggcttc aaggttggca tcaactacca
     4861 gcctcccact gtggtgcctg gtggagacct ggccaaggta cagagagctg tgtgcatgct
     4921 gagcaacacc acagccattg ctgaggcctg ggctcgcctg gaccacaagt ttgacctgat
     4981 gtatgccaag cgtgcctttg ttcactggta cgtgggtgag gggatggagg aaggcgagtt
     5041 ttcagaggcc cgtgaagata tggctgccct tgagaaggat tatgaggagg ttggtgtgga
     5101 ttctgttgaa ggagagggtg aggaagaagg agaggaatac taaggatcca ccggcctacg
     5161 tcgacgagct cactagtcgc ggccgctttc gaatctagag cctgcagtct cgacaagctt
     5221 gtcgagaagt actagaggat cataatcagc cataccacat ttgtagaggt tttacttgct
     5281 ttaaaaaacc tcccacacct ccccctgaac ctgaaacata aaatgaatgc aattgttgtt
     5341 gttaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     5401 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     5461 tcttatcatg tctggatctg atcactgctt gagcctagaa gatccggctg ctaacaaagc
     5521 ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa taactatcat aacccctagg
     5581 agatccgaac cagataagtg aaatctagtt ccaaactatt ttgtcatttt taattttcgt
     5641 attagcttac gacgctacac ccagttccca tctattttgt cactcttccc taaataatcc
     5701 ttaaaaactc catttccacc cctcccagtt cccaactatt ttgtccgccc acagcggggc
     5761 atttttcttc ctgttatgtt tttaatcaaa catcctgcca actccatgtg acaaaccgtc
     5821 atcttcggct actttttctc tgtcacagaa tgaaaatttt tctgtcatct cttcg
//
