LOCUS       sequence-351767-        4224 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  lox66-72 flanked gentamycin resistance cassette integrates into aquI
            integration site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4224)
  AUTHORS   Jones CM, Parrish S, Nielsen DR
  TITLE     Exploiting Polyploidy for Markerless and Plasmid-Free Genome 
            Engineering in Cyanobacteria.
  JOURNAL   ACS Synth Biol. 2021 Sep 17;10(9):2371-2382. doi: 
            10.1021/acssynbio.1c00269. Epub 2021 Aug 17.
  PUBMED    34530614
REFERENCE   2  (bases 1 to 4224)
  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"; doi: 
            "10.1021/acssynbio.1c00269"; journalName: "ACS Synth Biol"; date: 
            "2021-09-17- 17"; volume: "10"; issue: "9"; pages: "2371-2382"
COMMENT     Sequence Label: pCJ162
FEATURES             Location/Qualifiers
     source          1..4224
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(384..929)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(464..483)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1709..1742
                     /label=lox66
                     /bound_moiety="Cre recombinase"
                     /note="Right element (RE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    1743..1763
                     /label=BioBrick suffix
                     /note="universal suffix for all parts"
     CDS             1878..2411
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRNSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     terminator      complement(2462..2489)
                     /label=T7Te terminator
                     /note="phage T7 early transcription terminator"
     terminator      complement(2505..2576)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    2592..2625
                     /label=lox71
                     /bound_moiety="Cre recombinase"
                     /note="Left element (LE) mutant of loxP (Araki et al., 
                     2010). Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3205..3309
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3310..3378,3379..4170)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLKSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3528..3547)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
       61 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcgcgc
      121 tagcggagtg tatactggct tactatgttg gcactgatga gggtgtcagt gaagtgcttc
      181 atgtggcagg agaaaaaagg ctgcaccggt gcgtcagcag aatatgtgat acaggatata
      241 ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat
      301 ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta acagggaagt
      361 gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa
      421 atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt
      481 ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt
      541 ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt
      601 tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc
      661 cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc
      721 cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg
      781 aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg
      841 tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag
      901 agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt
      961 tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat
     1021 ataagttgta aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
     1081 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
     1141 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
     1201 ggaagcggaa gaagagtttc gcagaccata aaagatagga taaagaatca tggatattaa
     1261 aaacgttcat atcaaaaatc acgaacaaac agctcatgca ccttccactc tagaaaaaat
     1321 tcgtaaagtc aaacaagggg gtaaactctc agaacagaca aagacatttg gttcaaccta
     1381 ccgcaggtta gatccgaacc agccatctcc tacagtgacc cgtagtggtt atcgagattt
     1441 tattcatcct tttgaagatc gaatgctcac agttcgtgaa ctggcttgtt tgcaaacctt
     1501 tccccttgat tgggagttta ccggaactcg acttgattct tatagtagta aacgtaaagt
     1561 gacgatgact cagtttggac aagtgggtaa tgcagtaccg ccgttacttg ctgaagctgt
     1621 tgctaaagcg gttagcgaac agcttctgga tgtcattgat gaaaaataat cagtttgtaa
     1681 acaattccag tgaccttgtg acgactaata ccgttcgtat aatgtatgct atacgaagtt
     1741 atctgcagcg gccgctacta gtacaacaaa gccacgttgt gtctcaaaat ctctgatgtt
     1801 acattgcaca agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca
     1861 gtaatacaag gggtgttatg ttacgcaaca gcaacgatgt tacgcagcag ggcagtcgcc
     1921 ctaaaacaaa gttaggtggc tcaagtatgg gcatcattcg cacatgtagg ctcggccctg
     1981 accaagtcaa atccatgcgg gctgctcttg atcttttcgg tcgtgagttc ggagacgtag
     2041 ccacctactc ccaacatcag ccggactccg attacctcgg gaacttgctc cgtagtaaga
     2101 cattcatcgc gcttgctgcc ttcgaccaag aagcggttgt tggcgctctc gcggcttacg
     2161 ttctgcccaa gtttgagcag ccgcgtagtg agatctatat ctatgatctc gcagtctccg
     2221 gcgagcaccg gaggcagggc attgccaccg cgctcatcaa tctcctcaag catgaggcca
     2281 acgcgcttgg tgcttatgtg atctacgtgc aagcagatta cggtgacgat cccgcagtgg
     2341 ctctctatac aaagttgggc atacgggaag aagtgatgca ctttgatatc gacccaagta
     2401 ccgccaccta atcagaattg gttaattggt tgtaacactg gcagagcctc tagtatataa
     2461 acgcagaaag gcccacccga aggtgagcca gtgtgactct agtagagagc gttcaccgac
     2521 aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc
     2581 ctggctctag tataacttcg tataatgtat gctatacgaa cggtacttta caaaatcaaa
     2641 cccgatcgcc tctctatttt gataaatcta tgtctactcc ctctgttacc cctgtagaat
     2701 ctagcaccct aatcaaaacc cctgaactgc tggctccggc gggaaattgg gactgtgcga
     2761 tcaccgccgt ggagaatggg gctgatgcga tttattttgg gctggataaa tttaatgccc
     2821 ggatgcgatc acaaaacttt gtcgagtcag atttgccgga gttgatggca tacttacatc
     2881 ggcgcggcgt gaagggctat gtgacgttaa atacgctgat tttcacctcg gaattggcgg
     2941 cagtcgaaca atatttgcgg tcgattattg cggcgggagt cgatgcggcg atcgtccagg
     3001 atgtggggct gtgccaatta atttggcaat tgtcgcccga ttttccgatc catggttcga
     3061 cgcaaatgac cgtcaccagc gccgcagggg tcgagttcgc gcaaaacttg ggttgtgatt
     3121 tggtggatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca
     3181 ggtggcactt ttcggggaaa tgtgcgcgga acccccattt gtttattttt ctaaatacat
     3241 tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa
     3301 aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt
     3361 tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag
     3421 ttgggtgcac gagtgggtta catcgaactg gatctcaaaa gcggtaagat ccttgagagt
     3481 tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg
     3541 gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag
     3601 aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta
     3661 agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg
     3721 acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta
     3781 actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac
     3841 accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt
     3901 actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca
     3961 cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag
     4021 cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta
     4081 gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag
     4141 ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt
     4201 tagattgatt taaaacttca tttt
//
