LOCUS       sequence-492995-        4031 bp    DNA     circular SYN 18-NOV-2025
DEFINITION  for E.coli plasmid interference and PAM depletion assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 2776 to 2810)
  AUTHORS   Jin S, Zhu Z, Li Y, Zhang S, Liu Y, Li D, Li Y, Luo Y, Cheng Z, Zhao
            KT, Gao Q, Yang G, Li H, Liang R, Zhang R, Qiu JL, Zhang YE, Liu JG,
            Gao C
  TITLE     Functional RNA splitting drove the evolutionary emergence of type V 
            CRISPR-Cas systems from transposons.
  JOURNAL   Cell. 2025 Sep 29:S0092-8674(25)01035-9. doi: 
            10.1016/j.cell.2025.09.004.
  PUBMED    41027434
REFERENCE   2  (bases 1 to 4031)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2025 
            Sep 29:S0092-8674(25)01035-9. doi: 10.1016/j.cell.2025.09.004."
COMMENT     Sequence Label: pTarget-TTC
FEATURES             Location/Qualifiers
     source          1..4031
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(66..134,135..926)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(284..303)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1097..1684
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1585..1604
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1838..1855
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    1870..2009
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(1935..1954)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2095..2117
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2111..2302)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     terminator      complement(2551..2580)
                     /label=T3Te terminator
                     /note="phage T3 early transcription terminator"
     promoter        2776..2810
                     /label=﻿BBa_J23100
                     /note="Anderson constitutive promoter 
                     (https://parts.igem.org/Promoters/Catalog/Anderson). 
                     Measured strength relative to BBa_J223100: 1.0
                     "
                     /citation=[1]
     misc_RNA        2905..2954
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
     RBS             2986..2997
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             join(3004..3006,3007..3009,3010..3723)
                     /codon_start=1
                     /product="enhanced YFP"
                     /label=EYFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKLHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3049..3070)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3310..3329)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3657..3678
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     terminator      3736..3807
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3823..3850
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     misc_feature    3857..3877
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     terminator      3913..3944
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     promoter        join(3992..4031,1..65)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
       61 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
      121 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
      181 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
      241 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
      301 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
      361 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
      421 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
      481 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
      541 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
      601 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
      661 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
      721 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
      781 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
      841 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
      901 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
      961 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     1021 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     1081 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     1141 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     1201 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccga
     1261 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     1321 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     1381 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     1441 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     1501 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     1561 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     1621 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     1681 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     1741 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     1801 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     1861 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa
     1921 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc
     1981 tatcgctacg tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc
     2041 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc ttctccggga
     2101 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa
     2161 gctcatcagc gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct
     2221 cgttgagttt ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg
     2281 cggttttttc ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatccctg
     2341 aattcgcatc tagactgaac tggccgataa ttgcagacga acggatttac tttaccaagc
     2401 gagctcgtaa acttggtctg acagctctag ctccggcaaa aaaacgggca aggtgtcacc
     2461 accctgccct ttttctttaa aaccgaaaag attacttcgc gtttgccacc tgacgtctaa
     2521 gaaaaggaat attcagcaat ttgcccgtgc cgaagaaagg cccacccgtg aaggtgagcc
     2581 agtgagttga ttgctacgta attagttagt tagcccttag tgactcgaat tcgaattcct
     2641 cgtcagttcc gcttcgctac aggatcccta tctggagagt gcccgattgg gacccgactt
     2701 tcgcaggagc ctattacgga gccactgatg ctagcactgt accttgggtc ctttcctcac
     2761 gatagaagtt tcaggttgac ggctagctca gtcctaggta cagtgctagc atcagcagga
     2821 cgcactgacc actggatggt gatgtcaacg gtcataattt ttcgtgatcg acagcaacaa
     2881 gtgagcggac gcaactaatg gtagctgtca ccggatgtgc tttccggtct gatgagtccg
     2941 tgaggacgaa acagcctcta caaataattt tgtttaatac tagagaaaga ggagaaatac
     3001 tagatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     3061 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     3121 tacggcaagc tgaccctgaa gttcatctgc accacaggca agctgcccgt gccctggccc
     3181 accctcgtga ccaccttcgg ctacggcctg caatgcttcg cccgctaccc cgaccacatg
     3241 aagctgcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     3301 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     3361 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     3421 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     3481 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     3541 gccgaccact accagcagaa caccccaatc ggcgacggcc ccgtgctgct gcccgacaac
     3601 cactacctga gctaccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     3661 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     3721 taataggcca ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt
     3781 atctgttgtt tgtcggtgaa cgctctctac tagagtcaca ctggctcacc ttcgggtggg
     3841 cctttctgcg tttatatact agtagcggcc gctgcaggag tcactaaggg ttagttagtt
     3901 agattagcag aaagtcaaaa gcctccgacc ggaggctttt gactaaaact tcccttgggg
     3961 ttatcattgg ggctcactca aaggcggtaa tcgcggaacc cctatttgtt tatttttcta
     4021 aatacattca a
//
