LOCUS       pMAA166_LEU_inte        6662 bp DNA     circular SYN 24-AUG-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6662)
  AUTHORS   MIT
  TITLE     Direct Submission
  JOURNAL   Exported Dec 12, 2023 from SnapGene 7.1.0
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6662
                     /mol_type="other DNA"
                     /organism="recombinant plasmid"
     protein_bind    1..4
                     /label=BsaI
     misc_feature    20..162
                     /label=ConS
     misc_feature    20..22
                     /label=PAM
     misc_feature    complement(28..30)
                     /label=PAM
     primer_bind     42..61
                     /label=MAA_o109
     misc_feature    complement(57..59)
                     /label=PAM
     misc_feature    complement(63..65)
                     /label=PAM
     misc_feature    complement(119..121)
                     /label=PAM
     misc_feature    144..146
                     /label=PAM
     protein_bind    join(163..168,170..173)
                     /label=BsmBI
     misc_feature    170..194
                     /label=ConS scar
     misc_feature    176..178
                     /label=PAM
     primer_bind     195..226
                     /label=oENG111
     primer_bind     195..225
                     /label=oENG317_ytk
     protein_bind    195..198
                     /label=BsaI
     misc_feature    199..204
                     /label=spacer1_mut_A-->C
     promoter        205..218
                     /label=DBM_97-4_repeat_unit
                     /note="repeat this in the 5' direction to add additional 
                     43-8 binding sites
                     "
     misc_feature    205..215
                     /label=DBM_sTF_97-4
     misc_feature    216..218
                     /label=spacer2
     primer_bind     219..240
                     /label=oENG111
     primer_bind     219..239
                     /label=oENG317_ytk
     promoter        219..232
                     /label=DBM_97-4_repeat_unit
                     /note="repeat this in the 5' direction to add additional 
                     43-8 binding sites
                     "
     misc_feature    219..229
                     /label=DBM_sTF_97-4
     misc_feature    230..232
                     /label=spacer2
     primer_bind     233..254
                     /label=oENG111
     primer_bind     233..253
                     /label=oENG317_ytk
     promoter        233..246
                     /label=DBM_97-4_repeat_unit
                     /note="repeat this in the 5' direction to add additional 
                     43-8 binding sites
                     "
     misc_feature    233..243
                     /label=DBM_sTF_97-4
     misc_feature    244..246
                     /label=spacer2
     misc_feature    247..257
                     /label=DBM_sTF_97-4
     misc_feature    join(258,259..263)
                     /label=MAA_g33a
     misc_feature    join(258,259..263)
                     /label=MAA_g33b (amplify from pYTKA)
     misc_feature    join(258,259..263)
                     /label=spacer1
     primer_bind     263..281
                     /label=oENG110
     primer_bind     263..279
                     /label=oENG316_ytk
     promoter        264..277
                     /label=DBM_43-8_repeat_unit
                     /note="repeat this in the 5' direction to add additional 
                     43-8 binding sites
                     "
     protein_bind    264..274
                     /label=DBM_sTF43-8
                     /bound_moiety="sTF43-8"
     misc_feature    275..277
                     /label=spacer2
     primer_bind     278..295
                     /label=oENG110
     promoter        278..291
                     /label=DBM_43-8_repeat_unit
                     /note="repeat this in the 5' direction to add additional 
                     43-8 binding sites
                     "
     protein_bind    278..288
                     /label=DBM_sTF43-8
                     /bound_moiety="sTF43-8"
     misc_feature    289..291
                     /label=spacer2
     primer_bind     292..309
                     /label=oENG110
     primer_bind     292..307
                     /label=oENG316_ytk
     promoter        292..305
                     /label=DBM_43-8_repeat_unit
                     /note="repeat this in the 5' direction to add additional 
                     43-8 binding sites
                     "
     protein_bind    292..302
                     /label=DBM_sTF43-8
                     /bound_moiety="sTF43-8"
     misc_feature    303..305
                     /label=spacer2
     protein_bind    306..316
                     /label=DBM_sTF43-8
                     /bound_moiety="sTF43-8"
     primer_bind     317..336
                     /label=oENG314_ytk
     misc_feature    join(317,318..322)
                     /label=spacer1
     promoter        323..336
                     /label=DBM_42-10_repeat_unit
                     /note="repeat this in the 5' direction to add additional 
                     43-8 binding sites
                     "
     misc_feature    323..333
                     /label=DBM_sTF_42-10
     misc_feature    334..336
                     /label=spacer2_recode
     promoter        337..350
                     /label=DBM_42-10_repeat_unit
                     /note="repeat this in the 5' direction to add additional 
                     43-8 binding sites
                     "
     misc_feature    337..347
                     /label=DBM_sTF_42-10
     misc_feature    348..350
                     /label=spacer2_recode
     promoter        351..364
                     /label=DBM_42-10_repeat_unit
                     /note="repeat this in the 5' direction to add additional 
                     43-8 binding sites
                     "
     misc_feature    351..361
                     /label=DBM_sTF_42-10
     misc_feature    362..364
                     /label=spacer2
     misc_feature    365..375
                     /label=DBM_sTF_42-10
     misc_feature    376..381
                     /label=spacer3
     promoter        382..618
                     /label=miniCyc1_promoter
     primer_bind     complement(597..635)
                     /label=oENG315_ytk
     primer_bind     complement(601..631)
                     /label=oENG109
     misc_feature    619..624
                     /label=spacer4
     5'UTR           625..631
                     /label=kozak+start_codon
     primer_bind     631..653
                     /label=oENG518_ytk
     protein_bind    632..635
                     /label=_3
                     /label=BsaI
                     /bound_moiety="BsaI"
     CDS             633..1340
                     /codon_start=1
                     /product="Bright green-yellow fluorescent protein derived 
                     from Branchiostoma lanceolatum"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     primer_bind     633..660
                     /label=MA_065_gib
     primer_bind     633..653
                     /label=MA_o80_PKP40_seq
     primer_bind     721..739
                     /label=MAA_o228
     primer_bind     complement(824..842)
                     /label=MAA_o229
     primer_bind     929..948
                     /label=oENG520_seq
     primer_bind     complement(1314..1340)
                     /label=MA_o66_gib
     primer_bind     complement(1320..1347)
                     /label=oENG519_ytk
     primer_bind     complement(1320..1343)
                     /label=mNG_pGKL_R
     primer_bind     complement(1320..1340)
                     /label=MA_o87
     protein_bind    1343..1346
                     /label=BsaI
     terminator      1356..1580
                     /label=ScSSA1 Terminator
     protein_bind    1581..1584
                     /label=BsaI
     misc_feature    1585..1727
                     /label=ConE
     primer_bind     complement(1585..1604)
                     /label=MAA_o245
     protein_bind    join(1728..1733,1735..1738)
                     /label=BsmBI
     misc_feature    1735..1759
                     /label=ConE scar
     protein_bind    1775..1778
                     /label=BsaI
     promoter        1779..2426
                     /label=LEU2 Promoter
     primer_bind     complement(2386..2429)
                     /label=org349_leu2_tag_2
     CDS             2427..3521
                     /label=LEU2
     primer_bind     2430..2450
                     /label=org348_leu2_tag_1
     primer_bind     2430..2450
                     /label=org370_leu2_ttrz_f
     primer_bind     complement(3493..3521)
                     /label=org366_leu2_r_bamhi
     terminator      3522..3695
                     /label=AgLEU2 Terminator
     protein_bind    3696..3699
                     /label=BsaI
     misc_feature    3700..3719
                     /label=LEU2 3' barcode
     misc_feature    3720..4209
                     /label=LEU2 3' Homology
     protein_bind    4210..4213
                     /label=BsaI
     CDS             complement(4222..5232)
                     /label=SpecR
     promoter        complement(5233..5366)
                     /label=SpecR Promoter
     rep_origin      complement(5367..6130)
                     /direction=LEFT
                     /label=ColE1
     protein_bind    6139..6142
                     /label=BsaI
     misc_feature    6143..6642
                     /label=LEU2 5' Homology
     misc_feature    complement(6643..6662)
                     /label=LEU2 5' barcode
ORIGIN
        1 ccctgaattc gcatctagat ggtagagcca caaacagccg gtacaagcaa cgatctccag
       61 gaccatctga atcatgcgcg gatgacacga actcacgacg gcgatcacag acattaaccc
      121 acagtacaga cactgcgaca acgtggcaat tcgtcgcaat accgtctcac tgaactggcc
      181 gataattgca gacgaacggc attcattatg ggagattgat tatgggagat ggattatggg
      241 agatcgatta tgggagagct agcagagtga ggacttgaga gtgaggactg gagagtgagg
      301 actcgagagt gaggacgaat tcagacgctg ctcttcagac gctgctctgc agacgctgct
      361 ctccagacgc tgctcggatc ccagatccgc caggcgtgta tatatagcgt ggatggccag
      421 gcaactttag tgctgacaca tacaggcata tatatatgtg tgcgacgaca catgatcata
      481 tggcatgcat gtgctctgta tgtatataaa actcttgttt tcttcttttc tctaaatatt
      541 ctttccttat acattaggac ctttgcagca taaattacta tacttctata gacacacaaa
      601 cacaaataca cacactaatc tagatattaa atatggtttc aaagggtgaa gaggataaca
      661 tggcaagttt gccggccaca catgaacttc acattttcgg gagcataaat ggcgttgatt
      721 ttgacatggt cggtcagggg actgggaacc ccaacgacgg atacgaagaa ctcaacttga
      781 aatctaccaa aggtgatctc caattttcac cgtggattct tgttccgcac attgggtatg
      841 gatttcatca gtatttgcca taccctgatg gcatgtcacc cttccaggcc gctatggtcg
      901 atggtagcgg ctaccaggta caccgcacaa tgcagtttga agacggtgca tcactgaccg
      961 tgaactaccg atatacctat gagggttccc atattaaagg agaagctcaa gtaaagggaa
     1021 cgggattccc ggccgatggt cccgtgatga ctaactcctt gacagcagca gactggtgtc
     1081 gctctaagaa gacttatccc aacgataaaa ccattataag tacatttaaa tggagttata
     1141 caacgggcaa cggtaagagg tacagaagca cggcacggac cacttacacc ttcgctaagc
     1201 cgatggccgc aaactacttg aagaaccaac cgatgtacgt attccgaaag acggaactga
     1261 aacatagcaa aactgaactg aatttcaagg agtggcagaa agcgtttact gatgtcatgg
     1321 gtatggacga gctgtataag ggatcctaac tcgaggccaa ttggtgcggc aattgataat
     1381 aacgaaaatg tcttttaatg atctgggtat aatgaggaat tttccgaacg tttttacttt
     1441 atatatatat atacatgtaa catatattct atacgctata gagaaaggaa atttttcaat
     1501 taaaaaaaaa atagagaaag agtttcactt cttgattatc gctaacacta atggttgaag
     1561 tactgctact ttaattttat gctggaaatc tgctcgtcag tggtgctcac actgacgaat
     1621 catgtacaga tcataccgat gactgcctgg cgactcacaa ctaagcaaga cagccggaac
     1681 cagcgccggc gaacaccact gcatatatgg catatcacaa cagtccacgt ctcaagcagt
     1741 tacagagatg ttacgaacca ctagtgcact gcagtacatc gaggagaact tctagtatat
     1801 ctacatacct aatattattg ccttattaaa aatggaatcc caacaattac atcaaaatcc
     1861 acattctctt caaaatcaat tgtcctgtac ttccttgttc atgtgtgttc aaaaacgtta
     1921 tatttatagg ataattatac tctatttctc aacaagtaat tggttgtttg gccgagcggt
     1981 ctaaggcgcc tgattcaaga aatatcttga ccgcagttaa ctgtgggaat actcaggtat
     2041 cgtaagatgc aagagttcga atctcttagc aaccattatt tttttcctca acataacgag
     2101 aacacacagg ggcgctatcg cacagaatca aattcgatga ctggaaattt tttgttaatt
     2161 tcagaggtcg cctgacgcat ataccttttt caactgaaaa attgggagaa aaaggaaagg
     2221 tgagagcgcc ggaaccggct tttcatatag aatagagaag cgttcatgac taaatgcttg
     2281 catcacaata cttgaagttg acaatattat ttaaggacct attgtttttt ccaataggtg
     2341 gttagcaatc gtcttacttt ctaacttttc ttacctttta catttcagca atatatatat
     2401 atatatttca aggatatacc attctaatgt ctgcccctaa gaagatcgtc gttttgccag
     2461 gtgaccacgt tggtcaagaa atcacagccg aagccattaa ggttcttaaa gctatttctg
     2521 atgttcgttc caatgtcaag ttcgatttcg aaaatcattt aattggtggt gctgctatcg
     2581 atgctacagg tgttccactt ccagatgagg cgctggaagc ctccaagaag gctgatgccg
     2641 ttttgttagg tgctgtgggt ggtcctaaat ggggaaccgg tagtgttaga cctgaacaag
     2701 gtttactaaa aatccgtaaa gaacttcaat tgtacgccaa cttaagacca tgtaactttg
     2761 catccgactc tcttttagac ttatctccaa tcaagccaca atttgctaaa ggtactgact
     2821 tcgttgttgt cagagaatta gtgggaggta tttactttgg taagagaaag gaggacgatg
     2881 gtgatggtgt cgcttgggat agtgaacaat acaccgttcc agaagtgcaa agaatcacaa
     2941 gaatggccgc tttcatggcc ctacaacatg agccaccatt gcctatttgg tccttggata
     3001 aagctaatgt tttggcctct tcaagattat ggagaaaaac tgtggaggaa accatcaaga
     3061 acgaatttcc tacattgaag gttcaacatc aattgattga ttctgccgcc atgatcctag
     3121 ttaagaaccc aacccaccta aatggtatta taatcaccag caacatgttt ggtgatatca
     3181 tctccgatga agcctccgtt atcccaggtt ccttgggttt gttgccatct gcgtccttgg
     3241 cctctttgcc agacaagaac accgcatttg gtttgtacga accatgccac ggttctgctc
     3301 cagatttgcc aaagaataag gtcaacccta tcgccactat cttgtctgct gcaatgatgt
     3361 tgaaattgtc attgaacttg cctgaagaag gtaaggccat tgaagatgca gttaaaaagg
     3421 ttttggatgc aggtatcaga actggtgatt taggtggttc caacagtacc accgaagtcg
     3481 gtgatgctgt cgccgaagaa gttaagaaaa tccttgctta acctggagga cccttctctt
     3541 tagactattc tactcttatg cacgtaaaaa attctaggaa atatgtatta actaggagta
     3601 aaataaccgg ctagtggcat tcatatagcc gtctgtttac atctacatca cacatttcga
     3661 gtgtatatct cgcaacgttg gcgttaaata ggcaggagta ctcgtatcgc atgtcggtgc
     3721 gacacgaaat tacaaaatgg aatatgttca tagggtagac gaaactatat acgcaatcta
     3781 catacattta tcaagaagga gaaaaaggag gatgtaaagg aatacaggta agcaaattga
     3841 tactaatggc tcaacgtgat aaggaaaaag aattgcactt taacattaat attgacaagg
     3901 aggagggcac cacacaaaaa gttaggtgta acagaaaatc atgaaactat gattcctaat
     3961 ttatatattg gaggattttc tctaaaaaaa aaaaaataca acaaataaaa aacactcaat
     4021 gacctgacca tttgatggag tttaagtcaa taccttcttg aaccatttcc cataatggtg
     4081 aaagttccct caagaatttt actctgtcag aaacggcctt aacgacgtag tcgacctcct
     4141 cttcagtact aaatctacca ataccaaatc tgatggaaga atgggctaat gcatcatcct
     4201 tacccagcgc cgagcggccg cttatttgcc gactaccttg gtgatctcgc ctttcacgta
     4261 gtggacaaat tcttccaact gatctgcgcg cgaggccaag cgatcttctt cttgtccaag
     4321 ataagcctgt ctagcttcaa gtatgacggg ctgatactgg gccggcaggc gctccattgc
     4381 ccagtcggca gcgacatcct tcggcgcgat tttgccggtt actgcgctgt accaaatgcg
     4441 ggacaacgta agcactacat ttcgctcatc gccagcccag tcgggcggcg agttccatag
     4501 cgttaaggtt tcatttagcg cctcaaatag atcctgttca ggaaccggat caaagagttc
     4561 ctccgccgct ggacctacca aggcaacgct atgttctctt gcttttgtca gcaagatagc
     4621 cagatcaatg tcgatcgtgg ctggctcgaa gatacccgca agaatgtcat tgcgctgcca
     4681 ttctccaaat tgcagttcgc gcttagctgg ataacgccac ggaatgatgt cgtcgtgcac
     4741 aacaatggtg acttctacag cgcggagaat ctcgctctct ccaggggaag ccgaagtttc
     4801 caaaaggtcg ttgatcaaag ctcgccgcgt tgtttcatca agccttacgg tcaccgtaac
     4861 cagcaaatca atatcactgt gtggcttcag gccgccatcc actgcggagc cgtacaaatg
     4921 tacggccagc aacgtcggtt cgagatggcg ctcgatgacg ccaactacct ctgatagttg
     4981 agtcgatact tcggcgatca ccgcttccct cataatgttt aactttgttt tagggcgact
     5041 gccctgctgc gtaacatcgt tgctgctcca taacatcaaa catcgaccca cggcgtaacg
     5101 cgcttgctgc ttggatgccc gaggcataga ctgtacccca aaaaaacagt cataacaagc
     5161 catgaaaacc gccactgcgc cgttaccacc gctgcgttcg gtcaaggttc tggaccagtt
     5221 gcgtgagcgc atacgctact tgcattacag cttacgaacc gaacaggctt atgtccactg
     5281 ggttcgtgcc ttcatccgtt tccacggtgt gcgtcacccg gcaaccttgg gtagcagcga
     5341 agtcgaggca tttctgtcct ggctggtcat gaccaaaatc ccttaacgtg agttttcgtt
     5401 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     5461 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     5521 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5581 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5641 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5701 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5761 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5821 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5881 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5941 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     6001 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     6061 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     6121 ggataaccgt gcggccgcca atacccagaa gaacagtaaa ataaagcaag gtacgtgaaa
     6181 ttaatatttt taaatggttc taaccgatgc cgaagaactg cgcagtccgg ttataacgtc
     6241 tgacatgtcc ttttttgatt tggaatccaa ccactcaagt gactctgttc atttactttg
     6301 cgaaaaatat acccacaaat tgcccatcga aagtgaatcg caaaccacct tcagactggc
     6361 accgacaaag caaagattat acagacagag tactttatac gtaccgttaa gtctcaagca
     6421 aagggttttc ttatttactg aacgggtaaa gagtatctgg gccggcttgc caagatgcaa
     6481 accgaataag tatttcaaag ttgcatttgc cttagccgtc ctgacaccat tggctatttg
     6541 gatattttat attgactttc gtgtacattg atcacatcga ctgttctatt ggcaaatgaa
     6601 ccacgggcat tgactatttt tcaggttact actatatatt atcatcacgg gcaaggattg
     6661 ta
//
