LOCUS       Exported                3040 bp ds-DNA     circular SYN 13-MAY-2021
DEFINITION  Start-Stop Assembly Level 223 plasmid.
ACCESSION   .
VERSION     .
KEYWORDS    pStA223
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3040)
  AUTHORS   Taylor GM, Mordaka PM, Heap JT
  TITLE     Start-Stop Assembly: a functionally scarless DNA assembly system 
            optimized for metabolic engineering.
  JOURNAL   Nucleic Acids Res. 2018 Nov 20. pii: 5193345. doi: 
            10.1093/nar/gky1182.
  PUBMED    30462270
REFERENCE   2  (bases 1 to 3040)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3040
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(33..50)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(33..49)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(42..64)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(258..277)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     terminator      311..397
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     rep_origin      747..1292
                     /direction=RIGHT
                     /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     1193..1212
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1409..1426
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(1876..1895)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1887..2702
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1960..1979)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     terminator      2745..2839
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     protein_bind    2889..2910
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2925..2955
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2963..2979
                     /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     2968..2990
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2987..3003
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2987..3003
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             2999..282
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    3016..32
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
ORIGIN
        1 ctagaggatc cccgggtacc gagctcgaat tcactggccg tcgttttaca acgtcgtgac
       61 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
      121 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
      181 ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
      241 atatggtgca ctctcagtac aatctgctct gatgccgcat agtttccggt ctcatacttt
      301 acaagtcttc atttgtccta ctcaggagag cgttcaccga caaacaacag ataaaacgaa
      361 aggcccagtc tttcgactga gcctttcgtt ttatttgatg cccgaaaggg cctcgtgata
      421 cgcaaatctg gagccggtga gcgcggtatc attgcagcac tggggccaga tggtaagccc
      481 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga
      541 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac
      601 tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag
      661 atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg
      721 tcagacccct taataagatg atcttcttga gatcgttttg gtctgcgcgt aatctcttgc
      781 tctgaaaacg aaaaaaccgc cttgcagggc ggtttttcga aggttctctg agctaccaac
      841 tctttgaacc gaggtaactg gcttggagga gcgcagtcac caaaacttgt cctttcagtt
      901 tagccttaac cggcgcatga cttcaagact aactcctcta aatcaattac cagtggctgc
      961 tgccagtggt gcttttgcat gtctttccgg gttggactca agacgatagt taccggataa
     1021 ggcgcagcgg tcggactgaa cggggggttc gtgcatacag tccagcttgg agcgaactgc
     1081 ctacccggaa ctgagtgtca ggcgtggaat gagacaaacg cggccataac agcggaatga
     1141 caccggtaaa ccgaaaggca ggaacaggag agcgcacgag ggagccgcca gggggaaacg
     1201 cctggtatct ttatagtcct gtcgggtttc gccaccactg atttgagcgt cagatttcgt
     1261 gatgcttgtc aggggggcgg agcctatgga aaaacggctt tgccgcggcc ctctcacttc
     1321 cctgttaagt atcttcctgg catcttccag gaaatctccg ccccgttcgt aagccatttc
     1381 cgctcgccgc agtcgaacga ccgagcgtag cgagtcagtg agcgaggaag cggaatatat
     1441 cctgtatcac atattctgct gacgcaccgg tgcagccttt tttctcctgc cacatgaagc
     1501 acttcactga caccctcatc agtgccaaca tagtaagcca gtatacactc cgctagcgct
     1561 gaggtctgcc tcgtgaagaa ggtgttgctg actcatacca ggcctgaatc gccccatcat
     1621 ccagccagaa agtgagggag ccacggttga tgagagcttt gttgtaggtg gaccagttgg
     1681 tgattttgaa cttttgcttt gccacggaac ggtctgcgtt gtcgggaaga tgcgtgatct
     1741 gatccttcaa ctcagcaaaa gttcgattta ttcaacaaag ccacgttgtg tctcaaaatc
     1801 tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt
     1861 acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcga
     1921 ggccgcgatt aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata
     1981 atgtcgggca atcaggtgcg acaatctatc gattgtatgg gaagcccgat gcgccagagt
     2041 tgtttctgaa acatggcaaa ggtagcgttg ccaatgatgt tacagatgag atggtcagac
     2101 taaactggct gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg
     2161 atgatgcatg gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag
     2221 aatatcctga ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc
     2281 attcgattcc tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg
     2341 cgcaatcacg aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg
     2401 gctggcctgt tgaacaagtc tggaaagaaa tgcataagct tttgccattc tcaccggatt
     2461 cagtcgtcac tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa
     2521 taggttgtat tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc
     2581 tatggaactg cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg
     2641 gtattgataa tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct
     2701 aatcttgggg gaaattagcc gcagccgaac gaccgagcgc cttggactcc tgttgataga
     2761 tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt gccgccgggc
     2821 gttttttatt ggtgagaatg aagacaaact aggagagaga cccagtaggg cagtgagcgc
     2881 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     2941 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     3001 gaccatgatt acgccaagct tgcatgcctg caggtcgact
//
