LOCUS       Exported               14031 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Single-plasmid V. cholerae INTEGRATE system, encodes all proteins, 
            crRNA, and donor DNA. Entry vector encodes non-targeting crRNA, with
            BsaI sites for spacer cloning. pBBR1 backbone..
ACCESSION   .
VERSION     .
KEYWORDS    pSL1142 (pSPIN, pBBR1 backbone)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 14031)
  AUTHORS   Vo PLH, Ronda C, Klompe SE, Chen EE, Acree C, Wang HH, Sternberg SH
  TITLE     CRISPR RNA-guided integrases for high-efficiency, multiplexed 
            bacterial genome engineering.
  JOURNAL   Nat Biotechnol. 2020 Nov 23. pii: 10.1038/s41587-020-00745-y. doi: 
            10.1038/s41587-020-00745-y.
  PUBMED    33230293
REFERENCE   2  (bases 1 to 14031)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2020 Nov 23. pii: 10.1038/s41587-020-00745-y. doi: 
            10.1038/s41587-020-00745-y."
FEATURES             Location/Qualifiers
     source          1..14031
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      122..893
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             2373..3167
                     /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(2427..2446)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3037..3056
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     3611..3628
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3639..3673
                     /label=J23119 promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119)"
     CDS             10655..10666
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(12101..12119)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      12115..12162
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             12317..12976
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(12383..12402)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     primer_bind     complement(13176..13193)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(13176..13192)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(13185..13207)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(join(13490..14031,1..121))
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"