KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Bacterial Strains | ||
| One Shot™ PIR1 Chemically Competent E. coli | ThermoFischer | C101010 |
| TransforMax™ EC100D™ pir+ Electrocompetent E.coli | Lucigen | ECP09500 |
| BL21(DE3) Competent E. coli | New England Biolabs | C2527 |
| BL21(DE3) Electrocompetent Cells | Millipore Sigma | CMC0016 |
| Endura™ ElectroCompetent Cells | Lucigen | 60242 |
| One Shot™ Stbl3™ Chemically Competent E. coli | ThermoFischer | C737303 |
| BL21-CodonPlus (DE3)-RIPL Competent Cells | Agilent | 230280 |
| Trichormus variabilis | ATCC | 29413 |
| Chemicals, Peptides, and Recombinant Proteins | ||
| NEBNext® High-Fidelity 2X PCR Master Mix | New England Biolabs | M0541 |
| Q5® High-Fidelity 2X Master Mix | New England Biolabs | M0492 |
| KAPA HiFi HotStart ReadyMix | Roche | KK2602 |
| KOD Hot Start DNA Polymerase | Millipore Sigma | 71086-3 |
| E-Gel™ EX Agarose Gels, 1% | ThermoFischer | G401001 |
| E-Gel™ EX Agarose Gels, 2% | ThermoFischer | G401002 |
| E-Gel™ 48 Agarose Gels, 2% | ThermoFischer | G800802 |
| Wizard® SV Gel and PCR Clean-Up System | Promega | A9282 |
| Wizard® Genomic DNA Purification Kit | Promega | A1120 |
| QIAquick PCR Purification Kit | Qiagen | 28106 |
| QIAprep Spin Miniprep Kit | Qiagen | 27106 |
| PureYield™ Plasmid Midiprep System | Promega | A2495 |
| Gibson Assembly® Master Mix | New England Biolabs | E2611 |
| NEBuilder® HiFi DNA Assembly Master Mix | New England Biolabs | E2621 |
| Q5® Site-Directed Mutagenesis Kit | New England Biolabs | E0554 |
| FastDigest LguI | ThermoFischer | FD1934 |
| BsaI-HF®v2 | New England Biolabs | R3733 |
| BbsI-HF® | New England Biolabs | R3539 |
| T4 DNA Ligase | New England Biolabs | M0202 |
| IPTG | Goldbio | I2481C |
| TRI Reagent | ZYMO RESEARCH | R2050-1-200 |
| RNA Clean & Concentrator-25 | ZYMO RESEARCH | R1017 |
| DNase I (RNase-free) | New England Biolabs | M0303 |
| SUPERase•In™ RNase Inhibitor (20 U/μL) | ThermoFischer | AM2694 |
| Ribominus™ Transcriptome Isolation Kit | ThermoFischer | K155004 |
| Terminator™ 5′-Phosphate-Dependent Exonuclease | Lucigen | TER51020 |
| RiboGuard™ RNase Inhibitor | Lucigen | RG90910K |
| T4 Polynucleotide Kinase | New England Biolabs | M0201 |
| RNA 5′ Polyphosphatase | Lucigen | RP8092H |
| NEBNext® Small RNA Library Prep Set for Illumina® (Multiplex Compatible) | New England Biolabs | E7330 |
| TaqMan Fast Advanced Master Mix | ThermoFischer | 4444963 |
| ddPCR Supermix for Probes (No dUTP) | Bio-Rad | #1863024 |
| Droplet Generation Oil for Probes | Bio-Rad | #1863005 |
| FastDigest Eco32I | ThermoFischer | FD0304 |
| NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing | New England Biolabs | E7180S |
| Flow Cell (R9.4) | Oxford Nanopore Technologies | FLO-MIN106 |
| AMPure XP for PCR Purification | Beckman Coulter | A63881 |
| Tn5 | Schmid-Burgk et al., 2020. | N/A |
| cOmplete Protease Inhibitor Cocktail | Millipore sigma | 4693116001 |
| Strep-Tactin Superflow Plus | Qiagen | 30004 |
| D-Desthiobiotin | Millipore sigma | 71610-3 |
| Ulp1 SUMO protease | F. Zhang Lab | N/A |
| Amicon Ultra-15 Centrifugal Filter Units 30kDa NMWL | Millipore sigma | UFC903024 |
| Amicon Ultra-15 Centrifugal Filter Units 50kDa NMWL | Millipore sigma | UFC905024 |
| NuPAGE™ 4-12% Bis-Tris Protein Gels, 1.0 mm, 12-well | ThermoFischer | NP0322BOX |
| NuPAGE™ LDS Sample Buffer (4X) | ThermoFischer | NP0007 |
| Ampicillin, sodium salt | AmericanBio | Ab00115 |
| Carbenicillin disodium salt,89.0-100.5% anhydrous basis | Millipore sigma | C1389 |
| Spectinomycin dihydrochloride pentahydrate | Millipore sigma | S4014 |
| Kanamycin sulfate from Streptomyces kanamyceticus | Sigma | K4000 |
| Chloramphenicol | Sigma | C0378 |
| MiSeq Reagent Kits v2 | Illumina | MS-102 |
| NextSeq 500/550 High Output Kit v2, 75 cycles | Illumina | FC-404-2005 |
| Critical Commercial Assays | ||
| Qubit 1X dsDNA HS (High-Sensitivity) Assay Kit | ThermoFischer | Q33231 |
| eStain L1 Protein Staining System | GenScript | N/A |
| Deposited Data | ||
| Oligonucleotides | ||
| ctgtcgtcggtgacagattaatgtcattgtgac | IDT | Insert Probe |
| aacgctgatgggtcacgacg | IDT | T14 LE Reverse Primer |
| GACTGGAGTTCAGACGTGTGCTCTTCCGATCTtatctgcaaagtcgctgggg | IDT | PSP15 Target Forward Primer |
| GACTGGAGTTCAGACGTGTGCTCTTCCGATCTacgtacttcgccacctgaag | IDT | PSP42 Target Forward Primer |
| cgcggcaactttgtagtaccagc | IDT | E. coli Genome Forward Primer |
| cccttttcagatttctgcccgacgc | IDT | E. coli Genome Reverse Primer |
| acgttcgcgtttgccgtgcgtgtaatgtagtac | IDT | E. coli Genome Probe |
| gagcaagagattacgcgcagac | IDT | PAM library 6N upstream Primer |
| ctgcgtatgtgggcgctgcc | IDT | PAM library 6N downstream Primer |
| tgaaccgaattatatattgttgaacc | IDT | AvCAST RE Primer |
| taatggattcaaacaattgtttaaccg | IDT | AvCAST LE Primer |
| CTTTCCCTACACGACGCTCTTCCGATCTgagcaagagattacgcgcagac | IDT | PAM libarry 6N upstream NGS Primer |
| CTTTCCCTACACGACGCTCTTCCGATCTctgcgtatgtgggcgctgcc | IDT | PAM library 6N downstream NGS Primer |
| GACTGGAGTTCAGACGTGTGCTCTTCCGATCTcttttacttggtacaatcgtactcctttagagg | IDT | AvCAST RE NGS Primer |
| GACTGGAGTTCAGACGTGTGCTCTTCCGATCTcctttatcagtaatggattcaaacaattgtttaaccg | IDT | AvCAST LE NGS Primer |
| cgacagcatcgccagtcactatg | IDT | pTarget control forward Primer for ddPCR |
| caagtagcgaagcgagcaggac | IDT | pTarget contro reverse Primer for ddPCR |
| tgcgttgatgcaatttctatgcgcacccgt | IDT | pTarge control ddPCR Probe |
| agtcatttaataaggccactgttaaacg | IDT | PSP1 insertion site upstream for ddPCR |
| ctaccgcattaaagcttccgcc | IDT | PSP1 insertion site downstream Primer for ddPCR |
| cgacaacggcagattaaatattcgaactgt | IDT | AvCAST LE ddPCR Probe |
| ttcgaactgtaataattgtttaaccg | IDT | AvCAST-TTISS_LE 1st PCR Primer |
| gtctcgtgggctcggagatgtgtataagagacag | IDT | TTISS 1st PCR common Primer |
| AATGATACGGCGACCACCGAGATCTACACAAGTAGAGACACTCTTTCCCTACACGACGctcttccgatctTTGTTTAACCGAATTCTTTGTCTAC | IDT | AvCAST-TTISS_LE 2nd PCR Primer |
| caagcagaagacggcatacgagatcgatcatggtctcgtgggctcggagatgtgt | IDT | TTISS 2nd PCR comon Primer-1 |
| caagcagaagacggcatacgagattagatcctgtctcgtgggctcggagatgtgt | IDT | TTISS 2nd PCR comon Primer-2 |
| caagcagaagacggcatacgagatttactgtcgtctcgtgggctcggagatgtgt | IDT | TTISS 2nd PCR comon Primer-3 |
| caagcagaagacggcatacgagatggcatagggtctcgtgggctcggagatgtgt | IDT | TTISS 2nd PCR comon Primer-4 |
| caagcagaagacggcatacgagatcaaggcgagtctcgtgggctcggagatgtgt | IDT | TTISS 2nd PCR comon Primer-5 |
| caagcagaagacggcatacgagatgacgctatgtctcgtgggctcggagatgtgt | IDT | TTISS 2nd PCR comon Primer-6 |
| gcagaataaataaatcctggtgtc | IDT | pTarget glmS downstream Primer for ddPCR |
| CTTTCCCTACACGACGCTCTTCCGATCTagttggcagcatcacccgacgc | IDT | pTarget glmS downstream NGS primer |
| tggcaggatgtttgattaaaaac | IDT | Non-coding region between pstS and glmS ddPCR Primer |
| agcgataacatgcacatcatc | IDT | glmS Primer |
| CTTTCCCTACACGACGCTCTTCCGATCTgtgattgcaccaatcttctacaccgttc | IDT | glmS NGS Primer |
| GACTGGAGTTCAGACGTGTGCTCTTCCGATCTgcaaagttatggttccagtaacggc | IDT | PmcCAST RE NGS Primer |
| GACTGGAGTTCAGACGTGTGCTCTTCCGATCTgcataagcttgccgttgcgg | IDT | PmcCAST LE NGS Primer |
| gtaagctgttatatgtaaaactagaacagc | IDT | PmcCAST LE primer |
| CTTTCCCTACACGACGCTCTTCCGATCTagcttccttagctcctgaaaatctcg | IDT | pTarget tRNA-val upstream NGS Primer |
| tctggtactgcccagtagtg | IDT | tRNA-val primer for ddPCR |
| acccataactttgccgcaacggcaagctta | IDT | PmcCAST LE ddPCR Probe |
| CACTTTCATATCAGCATACTCCTGCTG | IDT | AvPSP2_Upstream Primer |
| CCCTCACCCCAGTCACTTACTTATG | IDT | AvPSP3_Upstream Primer |
| CTCACCCGGCAATTTCCCATTC | IDT | AvPSP4_Upstream Primer |
| TGTGAAGCTATGCGTTGCTGCC | IDT | AvPSP5_Upstream Primer |
| GATTAAATGTGACGCGACCCGC | IDT | AvPSP6_Upstream Primer |
| GTCCCAGTCCGATCTCTTGC | IDT | AvPSP7_Upstream Primer |
| CGGGCGGGATCGAGTATACAC | IDT | AvPSP8_Upstream Primer |
| TGCCTGACTTTGCTGCGCC | IDT | AvPSP9_Upstream Primer |
| TATCTGATGAGTTCCAAATTATGCCC | IDT | AvPSP10_Upstream Primer |
| ACCGTTGCATCATGTCGCCC | IDT | AvPSP11_Upstream Primer |
| ATTAGAAGGCGCACAATGTTACAGC | IDT | AvPSP12_Upstream Primer |
| CGACAGAACAACGATTGCCAGAG | IDT | AvPSP13_Upstream Primer |
| CTCCATTTTACCCATCCAGGGC | IDT | AvPSP14_Upstream Primer |
| AACGCTGCAACGTCCTGAGC | IDT | AvPSP15_Upstream Primer |
| CAGGTCGCCCGGATTACAGC | IDT | AvPSP16_Upstream Primer |
| CAGCTGGGCATTCGGTTGC | IDT | AvPSP17_Upstream Primer |
| ACGTCAGCATCCTAACAGCACC | IDT | AvPSP18_Upstream Primer |
| TATTAGTCTGAGTTATTTTTGTAGGGC | IDT | AvPSP19_Upstream Primer |
| GGACGTTAAAACGTCACGGCC | IDT | AvPSP20_Upstream Primer |
| GTCTGTGCGCTATGCCTATATTGG | IDT | AvPSP21_Upstream Primer |
| GAATTCGCTTTCTTGGAGAGCTTCTG | IDT | AvPSP22_Upstream Primer |
| ATCTGCAGGCTTTGCAGTCTGC | IDT | AvPSP23_Upstream Primer |
| TGAGTCCGCCCGGAACTCG | IDT | AvPSP24_Upstream Primer |
| GCTTAAGAATCGTAGAACCATCGGC | IDT | AvPSP25_Upstream Primer |
| GAGCCCGTGAACTGAAACCCTC | IDT | AvPSP26_Upstream Primer |
| CGACGGTGGTACGCATAACTTTC | IDT | AvPSP27_Upstream Primer |
| CGGTTTTACCCCTGTTACACGGG | IDT | AvPSP28_Upstream Primer |
| CTGCTAAAAATCGGCGCTAAGAACC | IDT | AvPSP29_Upstream Primer |
| CAGCTGAGACTGGATTTTTTCCAGC | IDT | AvPSP30_Upstream Primer |
| CTGTGCGGTGCCACAATCGG | IDT | AvPSP31_Upstream Primer |
| GCTGTTAGTCATGATTGGCCTGC | IDT | AvPSP32_Upstream Primer |
| CATCCGGCATTAAGCACCAACC | IDT | AvPSP33_Upstream Primer |
| GCAATGAACGATTATCCCTATCAAGC | IDT | AvPSP34_Upstream Primer |
| CAACCAGGCAACATGCCCGAC | IDT | AvPSP35_Upstream Primer |
| TGGCTATTCATTTGAAAGGAGGTCTG | IDT | AvPSP36_Upstream Primer |
| GTCATTCCGCTGCATATCATGAACGC | IDT | AvPSP37_Upstream Primer |
| TTGCATTCTGCGGGAAGGGATATC | IDT | AvPSP2_Downstream Primer |
| AGCGAATAGACAAATCGGTTGCCG | IDT | AvPSP3_Downstream Primer |
| GCCTGTGAACACACAGACAGAG | IDT | AvPSP4_Downstream Primer |
| GACTCTAACCGTCGGCCCC | IDT | AvPSP5_Downstream Primer |
| CTCATCGCCATTTACCTTCATGATAG | IDT | AvPSP6_Downstream Primer |
| GATCTGCGTTTTACAACTCAGATCAC | IDT | AvPSP7_Downstream Primer |
| CGGAAATCATACCATGAGGTTAATCC | IDT | AvPSP8_Downstream Primer |
| TGAACATAACGCCGATAGCAAAAGG | IDT | AvPSP9_Downstream Primer |
| TGGGCAAAATCAAGATACGCGCAG | IDT | AvPSP10_Downstream Primer |
| TAATTGCTAAGGAATAACCCAGTTGC | IDT | AvPSP11_Downstream Primer |
| CCTGGCCGTAAATGAAACACGC | IDT | AvPSP12_Downstream Primer |
| CTGAAAAAGTTTGTCCGCGATGC | IDT | AvPSP13_Downstream Primer |
| GCGCGGGGAACACTCTAAAC | IDT | AvPSP14_Downstream Primer |
| CGTAAGTTTCGCAGCTTATTAACAGC | IDT | AvPSP15_Downstream Primer |
| CCTTATCCGGCCTACAAAGTCG | IDT | AvPSP16_Downstream Primer |
| TTCTACGGGCAGGATGACTGG | IDT | AvPSP17_Downstream Primer |
| CGTGGGAAGGAGGCTATAATGG | IDT | AvPSP18_Downstream Primer |
| GCGTTCTGGTTGAATGGAACGC | IDT | AvPSP19_Downstream Primer |
| GTTGCCAGCAGGTATTATATCGCC | IDT | AvPSP20_Downstream Primer |
| GCAATGTTGCACCGTTTGCTGC | IDT | AvPSP21_Downstream Primer |
| GGTGGTTGACCTAAGGTAGCAG | IDT | AvPSP22_Downstream Primer |
| CCTTTCGCCCTGAATGCAGTC | IDT | AvPSP23_Downstream Primer |
| TCCGACAGTAAGCAAAAATTTGAGAC | IDT | AvPSP24_Downstream Primer |
| AGGTATTTAACCTGTGTTGATTGCTG | IDT | AvPSP25_Downstream Primer |
| ACGCAGCATTAATGCATAGTGGTTAAG | IDT | AvPSP26_Downstream Primer |
| GCGGGGCATTTTTCTTCCTGTTATG | IDT | AvPSP27_Downstream Primer |
| CACTAAACCGGGCCGTTTAGCC | IDT | AvPSP28_Downstream Primer |
| CATATTGCGCATGTTCGCGCAC | IDT | AvPSP29_Downstream Primer |
| GAAAAGCGCGCAAAGTGCGG | IDT | AvPSP30_Downstream Primer |
| GAATGCTTATACTGAAGACCGCGC | IDT | AvPSP31_Downstream Primer |
| CAAGAACAAGGCCATCCCTTTACC | IDT | AvPSP32_Downstream Primer |
| CCACTGTGTCGCTAAAAAGAGACAAC | IDT | AvPSP33_Downstream Primer |
| TTTGAGCCTGGCTTATCGCCG | IDT | AvPSP34_Downstream Primer |
| ATGATGCGGGTTCGATTCCCG | IDT | AvPSP35_Downstream Primer |
| CCGCCAGCTGAAGAAATCGCTAATTC | IDT | AvPSP36_Downstream Primer |
| CATCGGCTACGATGTAAAAATGGGTC | IDT | AvPSP37_Downstream Primer |
| Recombinant DNA | ||
| pHelper_ShCAST | Addgene | #127922 |
| pUC19 Vector | New England Biolabs | N3041 |
| pBluescript II SK (+) | Agilent | 212205 |
| pCDFDuet™-1 DNA | Millipore sigma | 71340 |
| pCOLADuet™-1 DNA | Millipore sigma | 71406 |
| pDonor(pSC101 origin)_ShCAST | F. Zhang Lab | N/A |
| pACYC 6N PAM library | Strecker et al., 2019. | N/A |
| pDonor_ShCAST_kanR | Addgene | #127924 |
| Software and Algorithms | ||
| Geneious | https://www.geneious.com/ | v2020 |
| QuantaSoft™ Software | Bio-Rad | #1864011 |
| Hmmer | http://hmmer.org/ | v3.1 |
| Blast+ | https://blast.ncbi.nlm.nih.gov/ | v2.9.0 |
| iTOL | https://itol.embl.de/ | N/A |
| MAFFT | https://mafft.cbrc.jp/ | V7.407 |
| Minced | https://github.com/ctSkennerton/minced | v0.4.2 |
| Prodigal | https://github.com/hyattpd/Prodigal | v2.6.3 |
| tRNA-ScanSE | http://lowelab.ucsc.edu/tRNAscan-SE/ | v2 |
| Infernal | http://eddylab.org/infernal/ | v1.1.3 |
| PyMOL | https://pymol.org/2/ | v1.2 |
| FastTree | http://www.microbesonline.org/fasttree/ | v2.1 |
| Hydrophobic Cluster Analysis | http://osbornite.impmc.upmc.fr/hca/hca-form.html | N/A |
| Other | ||
| Bench Protocol | (this paper) | STAR Methods |