LOCUS       pSYNMIBI_040_(pASPIre4)        5692 bp    DNA     circular UNA 11-NOV-2022
DEFINITION  pSH033 with new discriminator (pSH073).
ACCESSION   urn.local.mjeschek.3n-fja395h
VERSION     urn.local.mjeschek.3n-fja395h
KEYWORDS    .
SOURCE      
  ORGANISM  .
FEATURES             Location/Qualifiers
     att             21..46
                     /label="attB"
     att             47..70
                     /label="attB'"
     primer_bind     complement(70..89)
                     /%GC="50.0"
                     /Hairpin_Tm="42.7"
                     /Self_Dimer_Tm="None"
                     /Tm="56.5"
                     /Sequence="CCAAAGGTGACTAAGCGATG"
                     /Mismatches=0
                     /label="oSH158"
     Discriminator   71..220
                     /label="discriminator"
     primer_bind     82..98
                     /%GC="64.7"
                     /Hairpin_Tm="35.6"
                     /Self_Dimer_Tm="18.5"
                     /Tm="59.5"
                     /Sequence="ACCTTTGGGCCACGGTC"
                     /Mismatches=0
                     /label="oSH157"
     motif           complement(133..142)
                     /label="reading"
     motif           149..158
                     /label="reading"
     att             complement(221..244)
                     /label="attP'"
     att             complement(245..273)
                     /label="attP"
     primer_bind     complement(254..273)
                     /Tm_(binding_region)="58.3"
                     /Tm_(with_extension)="66.1"
                     /%GC_(binding_region)="50.0"
                     /%GC_(with_extension)="43.8"
                     /Hairpin_Tm_(with_extension)="46.1"
                     /Self_Dimer_Tm_(with_extension)="15.4"
                     /Sequence="TCGTGGTTTGTCTGGTCAAC"
                     /Mismatches=0
                     /label="oSH248"
     spacer          274..343
                     /label="spacer"
     promoter        350..467
                     /label="rhaB promoter"
     primer_bind     complement(458..479)
                     /Tm_(binding_region)="58.6"
                     /Tm_(with_extension)="66.1"
                     /%GC_(binding_region)="50.0"
                     /%GC_(with_extension)="40.0"
                     /Hairpin_Tm_(with_extension)="46.0"
                     /Self_Dimer_Tm_(with_extension)="15.3"
                     /Sequence="GAGCTCTTCATTACGACCAGTC"
                     /Mismatches=0
                     /label="oSM099"
     5'UTR           468..497
                     /label="5'UTR"
     RBS             498..514
                     /label="RBS_01"
     CDS             515..2020
                     /label="Bxb1"
     CDS             2021..2047
                     /label="GGS linker"
     CDS             2048..2764
                     /label="sfGFP"
     primer_bind     2584..2604
                     /%GC="33.3"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="None"
                     /Tm="50.2"
                     /Sequence="CCATTATCAACAAAATACTCC"
                     /Mismatches=0
                     /label="oSH249"
     CDS             2765..2797
                     /label="ssrA tag"
     CDS             2798..2803
                     /label="stop"
     motif           2849..2856
                     /label="PacI"
     terminator      2858..2962
                     /label="T1 terminator"
     regulatory      3129..3320
                     /label="ROP"
     regulatory      3422..3562
                     /label="bom"
     rep_origin      complement(3733..4352)
                     /label="pBR322_origin"
     rep_origin      4370..4615
                     /label="oriT"
     CDS             complement(4647..5462)
                     /label="KanR"
     terminator      complement(5590..5692)
                     /label="T0 terminator"
     primer_bind     5673..5692
                     /%GC="45.0"
                     /Hairpin_Tm="None"
                     /Self_Dimer_Tm="12.9"
                     /Tm="53.8"
                     /Sequence="TCTATCAACAGGAGTCCAAG"
                     /Mismatches=0
                     /label="oSH187"
ORIGIN      
        1 aagcttcaca tctgcagtaa tcggccggct tgtcgacgac ggcggtctcc gtcgtcagga
       61 tcatccgggc atcgcttagt cacctttggg ccacggtccg ctaccttaca ggaatagtac
      121 tcgtccttta atttggaatg aaccatggca gtcagttgtg ttgcgtttct tcgacctagt
      181 actcgctccc ttaggagaaa gacagatagc ttcttacccg gggtttgtac cgtacaccac
      241 tgagaccgcg gtggttgacc agacaaacca cgaaggttct gttaagtaac tgaacccaat
      301 gtcgttagtg acgcttacct cttaagaggt cactgaccta acaggatccc accacaattc
      361 agcaaattgt gaacatcatc acgttcatct ttccctggtt gccaatggcc cattttcctg
      421 tcagtaacga gaaggtcgcg aattcaggcg ctttttagac tggtcgtaat gaagagctca
      481 ataaatattt aatttatctc agaaaggcta agacatgcga gcactggttg ttattcgtct
      541 gagccgtgtt accgatgcaa ccactagtcc ggaacgtcag ctggaaagct gtcagcagct
      601 gtgtgcacag cgtggttggg atgttgttgg tgttgcagag gatctggatg ttagcggtgc
      661 agttgatccg tttgatcgta aacgtcgtcc gaatctggca cgttggctgg catttgaaga
      721 acagccgttt gatgttattg ttgcctatcg tgttgatcgt ctgacccgta gcattcgtca
      781 tctgcaacag ctggttcatt gggcagaaga tcataaaaaa ctggttgtga gcgcaaccga
      841 agcacatttt gataccacca ccccgtttgc agcagttgtt attgcactga tgggcaccgt
      901 tgcacagatg gaactggaag caattaaaga acgtaatcgt agcgcagccc attttaacat
      961 tcgtgcaggt aaatatcgtg gtagcctgcc tccgtggggt tatctgccga cgcgtgtaga
     1021 tggtgaatgg cgtctggttc ctgacccggt tcagcgtgaa cgtattctgg aagtatatca
     1081 tcgtgtggtg gataatcatg aaccgctgca tctggttgca catgatctga atcgtcgtgg
     1141 tgttctgagt cccaaagatt attttgctca gctgcaaggt cgtgaaccgc agggtcgtga
     1201 atggtctgca accgcactga aacgtagcat gattagcgaa gcaatgctgg gttatgcaac
     1261 cctgaatggt aaaaccgttc gtgatgatga tggtgcaccg ctggttcgtg cagaaccgat
     1321 tctgacacgt gaacagctgg aagcactgcg tgccgaactg gttaaaacca gccgtgcaaa
     1381 accggcagtt agcaccccga gcctgctgct gcgtgttctg ttttgtgcag tttgtggtga
     1441 accggcatac aaatttgccg gtggtggtcg taaacatccg cgttatcgtt gtcgtagcat
     1501 gggttttccg aaacattgtg gtaatggtac agttgcaatg gcagaatggg atgcattttg
     1561 cgaagaacag gttctggatc tgctgggtga tgccgaacgt ctggaaaaag tttgggttgc
     1621 aggtagcgat agcgcagttg aactggccga agttaatgcg gaactggtcg atctcaccag
     1681 tctgattgga agtcccgcat atcgtgcggg tagtcctcag cgtgaagcac tggatgcacg
     1741 tattgcagca ctggcagcac gtcaagaaga actggaaggt ctggaagcac gtccgagcgg
     1801 ttgggaatgg cgtgaaacag gtcagcgttt tggtgattgg tggcgtgagc aggataccgc
     1861 agcaaaaaat acctggctgc gtagtatgaa tgttcgcctg acctttgatg ttcgcggtgg
     1921 cctgacccgc accattgatt ttggcgatct gcaagaatat gaacagcatc tgcgtctggg
     1981 tagcgttgtt gaacgtctgc ataccggcat gagcaccggc ggtggcagcg gcggttctgg
     2041 tggctctagc aaaggagaag aacttttcac tggagttgtc ccaattcttg ttgaattaga
     2101 tggtgatgtt aatgggcaca aattttctgt ccgtggagag ggtgaaggtg atgctacaaa
     2161 cggaaaactc acccttaaat ttatttgcac tactggaaaa ctacctgttc cgtggccaac
     2221 acttgtcact actctgacct atggtgttca atgcttttcc cgttatccgg atcacatgaa
     2281 acggcatgac tttttcaaga gtgccatgcc cgaaggttat gtacaggaac gcactatatc
     2341 tttcaaagat gacgggacct acaagacgcg tgctgaagtc aagtttgaag gtgataccct
     2401 tgttaatcgt atcgagttaa agggtattga ttttaaagaa gatggaaaca ttcttggaca
     2461 caaactcgag tacaacttta actcacacaa tgtatacatc acggcagaca aacaaaagaa
     2521 tggaatcaaa gctaacttca aaattcgcca caacgttgaa gatggttccg ttcaactagc
     2581 agaccattat caacaaaata ctccaattgg cgatggccct gtccttttac cagacaacca
     2641 ttacctgtcg acacaatctg tcctttcgaa agatcccaac gaaaagcgtg accacatggt
     2701 ccttcttgag tttgtaactg ctgctgggat tacacatggc atggatgaac tctacaaaag
     2761 gcctgctgct aacgacgaaa actacgctct ggctgcttaa taagcggccg cggcctaggc
     2821 ggcctcctgt gtgaaattgt tatccgcttt aattaaaggc atcaaataaa acgaaaggct
     2881 cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt
     2941 aggacaaatc cgccgcccta gacagctggg cgcgcccccg ccgcatccat accgccagtt
     3001 gtttaccctc acaacgttcc agtaaccggg catgttcatc atcagtaacc cgtatcgtga
     3061 gcatcctctc tcgtttcatc ggtatcatta cccccatgaa cagaaatccc ccttacacgg
     3121 aggcatcagt gaccaaacag gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc
     3181 agacattaac gcttctggag aaactcaacg agctggacgc ggatgaacag gcagacatct
     3241 gtgaatcgct gcacgaccac gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg
     3301 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     3361 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     3421 gcgcagccat gacccagtca cgtagcgata gcggagtgta tactggctta actatgcggc
     3481 atcagagcag attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt
     3541 aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     3601 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     3661 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     3721 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     3781 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     3841 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     3901 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     3961 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4021 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4081 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4141 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg
     4201 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4261 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4321 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggccggcct acggccagcc
     4381 tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag tgaagaagga
     4441 acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc gttggataca
     4501 ccaaggaaag tctacacgaa ccctttggca aaatcctgta tatcgtgcga aaaaggatgg
     4561 atataccgaa aaaatcgcta taatgacccc gaagcagggt tatgcagcgg aaaaggacaa
     4621 cgcgcggacc gcggtccaat taattattag aaaaattcat ccagcatcag atgaaattgc
     4681 agtttgttca tatccggatt atcaatgcca tatttctgaa acagacgttt ttgcaggctc
     4741 gggctaaatt cgcccaggca gttccacaga atggccagat cctgataacg atccgcaatg
     4801 cccacacggc ccacatcaat gcagccaatc agtttgcctt catcgaaaat caggttatcc
     4861 aggctaaaat cgccgtgggt caccacgcta tccgggctaa acggcagcag tttatgcatt
     4921 tctttccaca cctgttccac cggccagccg ttacgttcat catcaaaatc gctcgcatcc
     4981 accaggccgt tgttcatacg gctctgcgcc tgggccagac gaaacacacg atcgctgtta
     5041 aacgggcagt tgcacaccgg aatgctatgc agacgacgca gaaacacggc cagcgcatcc
     5101 acaatgtttt cgccgctatc cggatattct tccagcacct gaaacgcggt tttgcccgga
     5161 atcgcggtgg tcagcagcca cgcatcatcc ggggtgcgaa taaaatgttt aatggtcggc
     5221 agcggcataa attcggtcag ccagttcaga cgcaccattt catcggtcac atcgttcgcc
     5281 acgctgcctt tgccatgttt cagaaacagt tccggcgcat ccggtttgcc atacagacga
     5341 taaatggtcg cgccgctctg acccacgtta tcacgcgccc atttatagcc atacagatcc
     5401 gcatccatgt tgctgttcag acgcggacgg ctacagctcg tttcacgctg aatatggctc
     5461 ataacacccc ttgtattact gtttatgtaa gcagacagtt ttattgttca tgatgatata
     5521 tttttatctt gtgcaatgta acatcagaga ttttgagaca caaatttaaa tcgtaattat
     5581 tggggacccc tggattctca ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca
     5641 aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca ag
//
