LOCUS       sequence-344746-        8023 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of I-SceI gene for genome engineering in Pseudomonas with
            sacB gene included for counterselection with sucrose on pBBR1-GmR 
            plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8023)
  AUTHORS   Kiattisewee C, Dong C, Fontana J, Sugianto W, Peralta-Yahya P, 
            Carothers JM, Zalatan JG
  TITLE     Portable bacterial CRISPR transcriptional activation enables 
            metabolic engineering in Pseudomonas putida.
  JOURNAL   Metab Eng. 2021 Apr 27. pii: S1096-7176(21)00063-X. doi: 
            10.1016/j.ymben.2021.04.002.
  PUBMED    33930546
REFERENCE   2  (bases 1 to 8023)
  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: "Metab Eng. 
            2021 Apr 27. pii: S1096-7176(21)00063-X. doi: 
            10.1016/j.ymben.2021.04.002."
COMMENT     Sequence Label: pCK255
FEATURES             Location/Qualifiers
     source          1..8023
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(354..359,360..376,377..382))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     terminator      complement(564..658)
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             complement(join(691..2025,2026..2112))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        complement(2113..2558)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     misc_feature    2816..2872
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     terminator      3684..3770
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    3904..3925
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3940..3945,3946..3963,3964..3970)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3978..3994
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3983..4005
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4002..4018
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4002..4018
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4014..4379
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWVPGPPSRSTVSISLISNSCSPGDPLV
                     LERPPPRWSSNSPYSESYYARSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEAR
                     TDRPSQQLRSLNGEWKL"
     primer_bind     4037..4057
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4039..4057
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     misc_feature    4070..4177
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     primer_bind     4086..4102
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     4087..4103
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4137..4153)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4137..4153)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4185..4204)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4186..4204)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4214..4231)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4214..4230)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4223..4245)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(4939..5601)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      5602..6373
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             complement(join(7655..8023,1..165))
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
ORIGIN
        1 ggctacgtct ccgaactcac gaccgaaaag atcaagagca gcccgcatgg atttgacttg
       61 gtcagggccg agcctacatg tgcgaatgat gcccatactt gagccaccta actttgtttt
      121 agggcgactg ccctgctgcg taacatcgtt gctgctgcgt aacatcgttg ctgctccata
      181 acatcaaaca tcgacccacg gcgtaacgcg cttgctgctt ggatgcccga ggcatagact
      241 gtacaaaaaa acagtcataa caagccatga aaaccgccac tgcgccgtta ccaccgctgc
      301 gttcggtcaa ggttctggac cagttgcgtg agcgcatacg ctacttgcat tacagtttac
      361 gaaccgaaca ggcttatgtc aactgggttc gtgccttcat ccgtttccac ggtgtgcgtc
      421 catgggcaaa tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca
      481 tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagttttt
      541 acgtaattat tggggacccc tggattctca ccaataaaaa acgcccggcg gcaaccgagc
      601 gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca
      661 agactagtat cggcattttc ttttgcgttt ttatttgtta actgttaatt gtccttgttc
      721 aaggatgctg tctttgacaa cagatgtttt cttgcctttg atgttcagca ggaagctagg
      781 cgcaaacgtt gattgtttgt ctgcgtagaa tcctctgttt gtcatatagc ttgtaatcac
      841 gacattgttt cctttcgctt gaggtacagc gaagtgtgag taagtaaagg ttacatcgtt
      901 aggatcaaga tccattttta acacaaggcc agttttgttc agcggcttgt atgggccagt
      961 taaagaatta gaaacataac caagcatgta aatatcgtta gacgtaatgc cgtcaatcgt
     1021 catttttgat ccgcgggagt cagtgaacag ataccatttg ccgttcattt taaagacgtt
     1081 cgcgcgttca atttcatctg ttactgtgtt agatgcaatc agcggtttca tcactttttt
     1141 cagtgtgtaa tcatcgttta gctcaatcat accgagagcg ccgtttgcta actcagccgt
     1201 gcgtttttta tcgctttgca gaagtttttg actttcttga cggaagaatg atgtgctttt
     1261 gccatagtat gctttgttaa ataaagattc ttcgccttgg tagccatctt cagttccagt
     1321 gtttgcttca aatactaagt atttgtggcc tttatcttct acgtagtgag gatctctcag
     1381 cgtatggttg tcgcctgagc tgtagttgcc ttcatcgatg aactgctgta cattttgata
     1441 cgtttttccg tcaccgtcaa agattgattt ataatcctct acaccgttga tgttcaaaga
     1501 gctgtctgat gctgatacgt taacttgtgc agttgtcagt gtttgtttgc cgtaatgttt
     1561 accggagaaa tcagtgtaga ataaacggat ttttccgtca gatgtaaatg tggctgaacc
     1621 tgaccattct tgtgtttggt cttttaggat agaatcattt gcatcgaatt tgtcgctgtc
     1681 tttaaagacg cggccagcgt ttttccagct gtcaatagaa gtttcgccga ctttttgata
     1741 gaacatgtaa atcgatgtgt catccgcatt tttaggatct ccggctaatg caaagacgat
     1801 gtggtagccg tgatagtttg cgacagtgcc gtcagcgttt tgtaatggcc agctgtccca
     1861 aacgtccagg ccttttgcag aagagatatt tttaattgtg gacgaatcga actcaggaac
     1921 ttgatatttt tcattttttt gctgttcagg gatttgcagc atatcatggc gtgtaatatg
     1981 ggaaatgccg tatgtttcct tatatggctt ttggttcgtt tctttcgcaa acgcttgagt
     2041 tgcgcctcct gccagcagtg cggtagtaaa ggttaatact gttgcttgtt ttgcaaactt
     2101 tttgatgttc atcgttcatg tctccttttt tatgtactgt gttagcggtc tgcttcttcc
     2161 agccctcctg tttgaagatg gcaagttagt tacgcacaat aaaaaaagac ctaaaatatg
     2221 taaggggtga cgccaaagta tacactttgc cctttacaca ttttaggtct tgcctgcttt
     2281 atcagtaaca aacccgcgcg atttactttt cgacctcatt ctattagact ctcgtttgga
     2341 ttgcaactgg tctattttcc tcttttgttt gatagaaaat cataaaagga tttgcagact
     2401 acgggcctaa agaactaaaa aatctatctg tttcttttca ttctctgtat tttttatagt
     2461 ttctgttgca tgggcataaa gttgcctttt taatcacaat tcagaaaata tcataatatc
     2521 tcatttcact aaataatagt gaacggcagg tatatgtgat gggttaaaaa ggatcgatcc
     2581 tctagaggat ccccctagag aataggaact tcggaatagg aacttcaaag cgtttccgaa
     2641 aacgagcgct tccgaaaatg caacgcgagc tgcgcacata cagctcactg ttcacgtcgc
     2701 acctatatct gcgtgttgcc tgtatatata tatacatgag aagaacggca tagtgcgtgt
     2761 ttatgcttaa atgcgtacct agtcgccagg gttttcccag tcacgacgcg gccgcaagct
     2821 tgcatgcctg caggtcgact ctagaggatc cccgggtacc gagctcgaat tcttattatt
     2881 tcaggaaagt ttcggaggag atagtgttcg gcagtttgta catcatctgc gggatcaggt
     2941 acggtttgat caggttgtag aagatcaggt aagacataga atcgatgtag atgatcggtt
     3001 tgtttttgtt gatttttacg taacagttca gttggaattt gttacgcaga cccttaacca
     3061 ggtattctac ttcttcgaaa gtgaaagact gggtgttcag tacgatcgat ttgttggtag
     3121 agtttttgtt gtaatcccat ttaccaccat catccatgaa ccagtatgcc agagacatcg
     3181 gggtcaggta gttttcaacc aggttgttcg ggatggtttt tttgttgtta acgatgaaca
     3241 ggttagccag tttgttgaaa gcctggtgtt tgaaagtctg ggcgccccag gtgattacca
     3301 ggttacccag gtggttaaca cgttcttttt tgtgcggcgg ggacagtacc cactgatcgt
     3361 acagcagaca tacgtggtcc atgtatgctt tgtttttcca ctcgaactgc atacagtagg
     3421 ttttaccttc atcacgagaa cggatgtaag catcacccag gatcagaccg atacctgctt
     3481 cgaactgttc gatgttcagt tcgatcagct gggatttgta ttctttcagc agtttagagt
     3541 tcggacccag gttcattacc tggttttttt tgatgttttt catatgttca tgactcccct
     3601 aggggtttag ttcctcacct tgtcgtatta tactatgccg atatactatg ccgatgatta
     3661 attgtcacat taattaaagg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     3721 ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgccct
     3781 agacagctgg gcgcgcctgc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     3841 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     3901 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc
     3961 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca
     4021 tgattacgcc aagcgcgcaa ttaaccctca ctaaagggaa caaaagctgg gtaccgggcc
     4081 ccccctcgag gtcgacggta tcgataagct tgatatcgaa ttcctgcagc ccgggggatc
     4141 cactagttct agagcggccg ccaccgcggt ggagctccaa ttcgccctat agtgagtcgt
     4201 attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta
     4261 cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg
     4321 cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg aaattgtaag
     4381 cgttaatatt ttgttaaaat ccgcgttaaa tttttgttaa atcagctcat tttttaacca
     4441 ataggccgta ctgcgatgag tggcagggcg gggcgtaatt tttttaaggc agttattggt
     4501 gcccttaaac gcctggttgc tacgcctgaa taagtgataa taagcggatg aatggcagaa
     4561 attcgaaagc aaattcgacc cggtcgtcgg ttcagggcag ggtcgttaaa tagccgctta
     4621 tgtctattgc tggtttaccg gtttattgac taccggaagc agtgtgaccg tgtgcttctc
     4681 aaatgcctga ggccagtttg ctcaggctct ccccgtggag gtaataattg acgatatgat
     4741 catttattct gcctcccaga gcctgataaa aacggtgaat ccgttagcga ggtgccgccg
     4801 gcttccattc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag
     4861 acaaggtata gggcggcgag gcggctacag ccgatagtct ggaacagcgc acttacgggt
     4921 tgctgcgcaa cccaagtgct accggcgcgg cagcgtgacc cgtgtcggcg gctccaacgg
     4981 ctcgccatcg tccagaaaac acggctcatc gggcatcggc aggcgctgct gcccgcgccg
     5041 ttcccattcc tccgtttcgg tcaaggctgg caggtctggt tccatgcccg gaatgccggg
     5101 ctggctgggc ggctcctcgc cggggccggt cggtagttgc tgctcgcccg gatacagggt
     5161 cgggatgcgg cgcaggtcgc catgccccaa cagcgattcg tcctggtcgt cgtgatcaac
     5221 caccacggcg gcactgaaca ccgacaggcg caactggtcg cggggctggc cccacgccac
     5281 gcggtcattg accacgtagg ccgacacggt gccggggccg ttgagcttca cgacggagat
     5341 ccagcgctcg gccaccaagt ccttgactgc gtattggacc gtccgcaaag aacgtccgat
     5401 gagcttggaa agtgtcttct ggctgaccac cacggcgttc tggtggccca tctgcgccac
     5461 gaggtgatgc agcagcattg ccgccgtggg tttcctcgca ataagcccgg cccacgcctc
     5521 atgcgctttg cgttccgttt gcacccagtg accgggcttg ttcttggctt gaatgccgat
     5581 ttctctggac tgcgtggcca tgcttatctc catgcggtag gggtgccgca cggttgcggc
     5641 accatgcgca atcagctgca acttttcggc agcgcgacaa caattatgcg ttgcgtaaaa
     5701 gtggcagtca attacagatt ttctttaacc tacgcaatga gctattgcgg ggggtgccgc
     5761 aatgagctgt tgcgtacccc ccttttttaa gttgttgatt tttaagtctt tcgcatttcg
     5821 ccctatatct agttctttgg tgcccaaaga agggcacccc tgcggggttc ccccacgcct
     5881 tcggcgcggc tccccctccg gcaaaaagtg gcccctccgg ggcttgttga tcgactgcgc
     5941 ggccttcggc cttgcccaag gtggcgctgc ccccttggaa cccccgcact cgccgccgtg
     6001 aggctcgggg ggcaggcggg cgggcttcgc ccttcgactg cccccactcg cataggcttg
     6061 ggtcgttcca ggcgcgtcaa ggccaagccg ctgcgcggtc gctgcgcgag ccttgacccg
     6121 ccttccactt ggtgtccaac cggcaagcga agcgcgcagg ccgcaggccg gaggcttttc
     6181 cccagagaaa attaaaaaaa ttgatggggc aaggccgcag gccgcgcagt tggagccggt
     6241 gggtatgtgg tcgaaggctg ggtagccggt gggcaatccc tgtggtcaag ctcgtgggca
     6301 ggcgcagcct gtccatcagc ttgtccagca gggttgtcca cgggccgagc gaagcgagcc
     6361 agccggtggc cgctcgcggc catcgtccac atatccacgg gctggcaagg gagcgcagcg
     6421 accgcgcagg gcgaagcccg gagagcaagc ccgtagggcg ccgcagccgc cgtaggcggt
     6481 cacgactttg cgaagcaaag tctagtgagt atactcaagc attgagtggc ccgccggagg
     6541 caccgccttg cgctgccccc gtcgagccgg ttggacacca aaagggaggg gcaggcatgg
     6601 cggcatacgc gatcatgcga tgcaagaagc tggcgaaaat gggcaacgtg gcggccagtc
     6661 tcaagcacgc ctaccgcgag cgcgagacgc ccaacgctga cgccagcagg acgccagaga
     6721 acgagcactg ggcggccagc agcaccgatg aagcgatggg ccgactgcgc gagttgctgc
     6781 cagagaagcg gcgcaaggac gctgtgttgg cggtcgagta cgtcatgacg gccagcccgg
     6841 aatggtggaa gtcggccagc caagaacagc aggcggcgtt cttcgagaag gcgcacaagt
     6901 ggctggcgga caagtacggg gcggatcgca tcgtgacggc cagcatccac cgtgacgaaa
     6961 ccagcccgca catgaccgcg ttcgtggtgc cgctgacgca ggacggcagg ctgtcggcca
     7021 aggagttcat cggcaacaaa gcgcagatga cccgcgacca gaccacgttt gcggccgctg
     7081 tggccgatct agggctgcaa cggggcatcg agggcagcaa ggcacgtcac acgcgcattc
     7141 aggcgttcta cgaggccctg gagcggccac cagtgggcca cgtcaccatc agcccgcaag
     7201 cggtcgagcc acgcgcctat gcaccgcagg gattggccga aaagctggga atctcaaagc
     7261 gcgttgagac gccggaagcc gtggccgacc ggctgacaaa agcggttcgg caggggtatg
     7321 agcctgccct acaggccgcc gcaggagcgc gtgagatgcg caagaaggcc gatcaagccc
     7381 aagagacggc ccgagacctt cgggagcgcc tgaagcccgt tctggacgcc ctggggccgt
     7441 tgaatcggga tatgcaggcc aaggccgccg cgatcatcaa ggccgtgggc gaaaagctgc
     7501 tgacggaaca gcgggaagtc cagcgccaga aacaggccca gcgccagcag gaacgcgggc
     7561 gcgcacattt ccccgaaaag tgccacctgc ggcgttgtga caatttaccg aacaactccg
     7621 cggccgggaa gccgatctcg gcttgaacga attgttaggt ggcggtactt gggtcgatat
     7681 caaagtgcat cacttcttcc cgtatgccca actttgtata gagagccact gcgggatcgt
     7741 caccgtaatc tgcttgcacg tagatcacat aagcaccaag cgcgttggcc tcatgcttga
     7801 ggagattgat gagcgcggtg gcaatgccct gcctccggtg ctctccggag actgcgagat
     7861 catagatata gatctcacta cgcggctgct caaacttggg cagaacgtaa gccgcgagag
     7921 cgccaacaac cgcttcttgg tcgaaggcag caagcgcgat gaatgtctta ctacggagca
     7981 agttcccgag gtaatcggag tccggctgat gttgggagta ggt
//
