LOCUS       Exported                5305 bp ds-DNA     circular SYN 19-NOV-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5305)
  AUTHORS   Reuben Philip
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Jun 12, 2024 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5305
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     CDS             complement(146..396)
                     /codon_start=1
                     /gene="lacZ"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="LAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQL
                     RSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICSDAA"
     primer_bind     328..352
                     /label=pREPAIR-LHA Seq
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    396
                     /label=MCS
                     /note="pUC19 multiple cloning site"
     misc_feature    402..474
                     /label=73bp random stuffer - blast safe
     misc_feature    475..1011
                     /label=TSC22D2-N-LHA-537bp
     primer_bind     500..548
                     /label=82% GC
     CDS             1012..1032
                     /codon_start=1
                     /product="Nterm. To allow for easy and universal switching 
                     of resistances with a primer. Allow for universal Gibson 
                     homology for HDR fragments."
                     /label=Universal seq
                     /translation="GKSKTGS"
     primer_bind     1012..1032
                     /label=N-Cassette (Fw) - LP7886
     CDS             1033..1428
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=Blast
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             1438..1494
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     1438..1462
                     /label=P2A (Fw) - LP7890
     primer_bind     complement(1474..1494)
                     /label=P2A (Rev) - LP7891
     CDS             1501..1527
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1531..1557
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1567..2343
                     /codon_start=1
                     /product="N-terminally truncated mutant of the E. coli 
                     biotin ligase BioID engineered for improved catalytic 
                     efficiency (Branon et al., 2018)"
                     /label=miniTurbo
                     /translation="MAIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDA
                     CIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAIGLGPVIGIVMAEALRKLGA
                     DKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWIT
                     LQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKE
                     IFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK"
     regulatory      1567..1570
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     complement(2008..2020)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             2344..2370
                     /codon_start=1
                     /product="Linker includes cut site to allow for editing 
                     once arm insertion and final repair vectors are made. "
                     /label=High-Flex Linker
                     /translation="GGSGGSGGT"
     primer_bind     complement(2352..2370)
                     /label=N-Cassette (Rev) - LP7887
     misc_feature    2371..3065
                     /label=N-TSC22D2 RHA
     CDS             complement(3067..3088)
                     /codon_start=1
                     /gene="lacZ"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPS"
     misc_feature    3067..3071
                     /label=MCS
                     /note="pUC19 multiple cloning site"
     primer_bind     complement(3084..3100)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3108..3124
                     /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     complement(3121..3144)
                     /label=pREPAIR-RHA Seq
     promoter        complement(3132..3162)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3315..3332)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3486..4074)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3566..3585)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4245..5105)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4868..4887
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5106..5210)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ctcattttca atatacaagc
      421 aacccaagaa ggctgagcct agactcaact ggttgctgag taaactccag actttccttc
      481 cctcagtcag agagcccagc gctgacgccg gcaccggcct gaggagcccg agcggggcag
      541 caccgcccct cacgccgccg ccaccgcctc ctcctcctcc tcttcgccct cccactccca
      601 cccccagcca aggcctgctg accgcgccga gcgccgagct ggactgacca cggctgcccg
      661 gagacgagag aggaagcagc cggcccgccc ctgggttcgc gctctcgccg cctctgaggg
      721 aattgaattg aggcgccgcg gctgcgagag ctaaaaagga aggaggagcc gccgcgggac
      781 tgagacgggg gcagagccga agagaccgac acagagaagg aaacgaggag gaggatgtct
      841 caccgggcgg ccagcgcctg gatcagcccg tgactcttaa cagcggcggg cctcagaccc
      901 cagcgcagac tcggactttg tctttggggg cccgtgctct gccctccccg gtttccgaca
      961 ggacccagag gagccggcgt gcctctctgc cctccagcct tcttcaccat gggcaagagc
     1021 aagaccggta gcatggccaa gcctttgtct caagaagaat ccaccctcat tgaaagagca
     1081 acggctacaa tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc
     1141 tctagcgacg gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt
     1201 gcagaactcg tggtgctggg cactgctgct gctgcggcag ctggcaacct gacttgtatc
     1261 gtcgcgatcg gaaatgagaa caggggcatc ttgagcccct gcggacggtg ccgacaggtg
     1321 cttctcgatc tgcatcctgg gatcaaagcc atagtgaagg acagtgatgg acagccgacg
     1381 gcagttggga ttcgtgaatt gctgccctct ggttatgtgt gggagggcgg atccggagct
     1441 actaacttca gcctgctgaa gcaggccgga gacgtggagg agaaccctgg accttctaga
     1501 tacccctacg acgtgcccga ctacgccggc tatccgtatg atgtcccgga ctatgcagga
     1561 agcggaatgg cgatcccgct gctgaacgct aaacagattc tgggacagct ggacggcggg
     1621 agcgtggcag tcctgcctgt ggtcgactcc accaatcagt acctgctgga tcgaatcggc
     1681 gagctgaaga gtggggatgc ttgcattgca gaatatcagc aggcagggag aggaagcaga
     1741 gggaggaaat ggttctctcc ttttggagct aacctgtacc tgagtatgtt ttggcgcctg
     1801 aagcggggac cagcagcaat cggcctgggc ccggtcatcg gaattgtcat ggcagaagcg
     1861 ctgcgaaagc tgggagcaga caaggtgcga gtcaaatggc ccaatgacct gtatctgcag
     1921 gatagaaagc tggcaggcat cctggtggag ctggccggaa taacaggcga tgctgcacag
     1981 atcgtcattg gcgccgggat taacgtggct atgaggcgcg tggaggaaag cgtggtcaat
     2041 cagggctgga tcacactgca ggaagcaggg attaacctgg acaggaatac tctggccgct
     2101 atgctgatcc gagagctgcg ggcagccctg gaactgttcg agcaggaagg cctggctcca
     2161 tatctgtcac ggtgggagaa gctggataac ttcatcaata gacccgtgaa gctgatcatt
     2221 ggggacaaag agattttcgg gattagccgg gggattgata aacagggagc cctgctgctg
     2281 gaacaggacg gagttatcaa accctggatg ggcggagaaa tcagtctgcg gtctgccgaa
     2341 aagggaggaa gcggaggatc tggcggtacc tccaagatgc cggccaagaa gaagagctgc
     2401 ttccagatca ccagtgtcac cacggcccag gtggccacta gcatcaccga ggacaccgag
     2461 agcttggacg acccggacga gtcacgcaca gaggacgtct cctccgagat tttcgacgtc
     2521 tctcgggcca cggattatgg ccctgaggag gtctgcgagc gcagctcttc cgaagagacg
     2581 cttaacaatg ttggggatgc ggagactccc gggaccgtct ccccaaacct cctcctagat
     2641 gggcagctgg cagcggcggc tgctgctccc gccaacggag gaggagtcgt ttcggcccgg
     2701 agcgtgtctg gggcgctcgc cagtaccctg gcggcggctg ccacttcggc ccccgccccc
     2761 ggagcacccg gcggccccca gctcgcgggc tcatccgccg ggccagtgac tgcagcccca
     2821 tctcagcctc ccaccacatg tagttcccgt tttcgcgtga tcaagctgga ccacgggagc
     2881 ggagagccct atagacgcgg ccgatggacg tgtatggaat actatgagag ggattcagac
     2941 agcagcgtcc tgactagatc cggggattgc attagacaca gcagtacttt tgaccagact
     3001 gcggagcggg acagcggcct gggcgccacc ggagggtcgg tggtggtagt agtggcctcc
     3061 atgcaaagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     3121 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     3181 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     3241 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     3301 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     3361 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     3421 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     3481 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     3541 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     3601 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     3661 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     3721 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     3781 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     3841 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     3901 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     3961 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     4021 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     4081 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     4141 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     4201 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     4261 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     4321 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     4381 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     4441 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     4501 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     4561 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     4621 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4681 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4741 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4801 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4861 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4921 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4981 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     5041 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     5101 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     5161 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     5221 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     5281 aggcgtatca cgaggccctt tcgtc
//
