LOCUS       Exported                5228 bp ds-DNA     circular SYN 16-AUG-2022
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5228)
  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..5228
                     /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
     misc_feature    377..401
                     /label=GGIBSON-LHA-A
     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..895
                     /label=TSC1-N-LHA
     CDS             896..916
                     /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     896..916
                     /label=N-Cassette (Fw) - LP7886
     CDS             917..1513
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=Puro
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     CDS             1514..1522
                     /codon_start=1
                     /label=GSG-2A
                     /note="N-terminal GSG for efficient P2A transition. 
                     Modified to include BamHI site. "
                     /translation="GSG"
     CDS             1523..1579
                     /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     1523..1547
                     /label=P2A (Fw) - LP7890
     primer_bind     complement(1559..1579)
                     /label=P2A (Rev) - LP7891
     CDS             1586..1612
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1616..1642
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1652..2359
                     /codon_start=1
                     /product="bright monomeric yellow-green fluorescent protein
                     derived from LanYFP (Shaner et al., 2013)"
                     /label=mNeonGreen
                     /translation="MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYE
                     ELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFED
                     GASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIIS
                     TFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQ
                     KAFTDVMGMDELYK"
     CDS             2360..2386
                     /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(2368..2386)
                     /label=N-Cassette (Rev) - LP7887
     misc_feature    2387..2988
                     /label=TSC1-N-RHA
     misc_feature    2989..3013
                     /label=GIBSON-RHA-B
     CDS             complement(2990..3011)
                     /codon_start=1
                     /gene="lacZ"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPS"
     misc_feature    2990..2994
                     /label=MCS
                     /note="pUC19 multiple cloning site"
     primer_bind     complement(3007..3023)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3031..3047
                     /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(3044..3067)
                     /label=pREPAIR-RHA Seq
     promoter        complement(3055..3085)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3238..3255)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3409..3997)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3489..3508)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(4168..5028)
                     /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     4791..4810
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5029..5133)
                     /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 cccgggggtg gcgaggggaa
      421 ggcgacattt gcttggcgct gcctccgtgc agagcggccg ggagggcttt gcagcggccg
      481 ccgacgcggc gggaggaggg ggctgaggcg gcttcggagt tggccggaga aggtgggcat
      541 ttctcgcttt tcctcccctt cctgcgctct ccccctccct cctgccccgc accccacgtg
      601 aagcagaata taaagggggg tgtgaggcta gaggggaaag tgaatggcga aggactgaag
      661 ggatcccccc ttcgggtccc cggccgccct gttcaccctc gttcatcctc ctttccgaag
      721 ctcgctctcg aaggcaggag cgaccggcgc ctttggctga ggaggaggag aaggaggaat
      781 cgcgccaggc ggagcgtcag gtcccgtttt cctctccggc gtctccaata caaagattac
      841 ggtgcagaag gaaattgcac tcgtctcctc cgcgcccccg gtacccaaca caatgggcaa
      901 gagcaagacc ggtagcatga ccgagtacaa gcccacggtg cgcctcgcca cccgcgacga
      961 cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc gactaccccg ccacgcgcca
     1021 caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag ctgcaagaac tcttcctcac
     1081 gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac gacggcgccg cggtggcggt
     1141 ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc gccgagatcg gcccgcgcat
     1201 ggccgagttg agcggttccc ggctggccgc gcagcaacag atggaaggcc tcctggcgcc
     1261 gcaccggccc aaggagcccg cgtggttcct ggccaccgtc ggcgtctcgc ccgaccacca
     1321 gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg gaggcggccg agcgcgccgg
     1381 ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc cccttctacg agcggctcgg
     1441 cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg cgcacctggt gcatgacccg
     1501 caagcccggt gccggatccg gagctactaa cttcagcctg ctgaagcagg ccggagacgt
     1561 ggaggagaac cctggacctt ctagataccc ctacgacgtg cccgactacg ccggctatcc
     1621 gtatgatgtc ccggactatg caggaagcgg aatggtgagc aagggcgagg aggataacat
     1681 ggcctctctc ccagcgacac atgagttaca catctttggc tccatcaacg gtgtggactt
     1741 tgacatggtg ggtcagggca ccggcaatcc aaatgatggt tatgaggagt taaacctgaa
     1801 gtccaccaag ggtgacctcc agttctcccc ctggattctg gtccctcata tcgggtatgg
     1861 cttccatcag tacctgccct accctgacgg gatgtcgcct ttccaggccg ccatggtaga
     1921 tggctccgga taccaagtcc atcgcacaat gcagtttgaa gatggtgcct cccttactgt
     1981 taactaccgc tacacctacg agggaagcca catcaaagga gaggcccagg tgaaggggac
     2041 tggtttccct gctgacggtc ctgtgatgac caactcgctg accgctgcgg actggtgcag
     2101 gtcgaagaag acttacccca acgacaaaac catcatcagt acctttaagt ggagttacac
     2161 cactggaaat ggcaagcgct accggagcac tgcgcggacc acctacacct ttgccaagcc
     2221 aatggcggct aactatctga agaaccagcc gatgtacgtg ttccgtaaga cggagctcaa
     2281 gcactccaag accgagctca acttcaagga gtggcaaaag gcctttaccg atgtgatggg
     2341 catggacgag ctgtacaagg gaggaagcgg aggatctggc ggtacccacc agccgcctga
     2401 gtccaccgcc gcggccgccg ccgctgcaga cattagcgct aggaagatgg cgcacccggc
     2461 aatgttccct cgaaggggca gcggtagtgg cagcgcctct gctctcaatg cagcaggtac
     2521 cggcgtcggt agtaatgcca catcttccga ggattttccg cctccgtcgc tgcttcagcc
     2581 gccgccccct gcagcatctt ctacgtcggg accacagcct ccgcctccac aaagcctgaa
     2641 cctcctttcg caggctcagc tgcaggcaca gcctcttgcg ccaggcggaa ctcaaatgaa
     2701 aaagaaaagt ggcttccaga taactagcgt tactcctgct cagatctccg ctagtatcag
     2761 ctctaacaac agtatagcag aggacactga gagctatgat gatctggatg aatctcacac
     2821 ggaagatctc tcttcttcgg agatccttga tgtgtcactt tccagggcta ctgacttagg
     2881 ggagcccgaa cgcagctcct cagaagagac cctaaataac ttccaggaag ccgagacacc
     2941 tggggcagtc tctcccaacc agccccacct tcctcagcct catttgccaa gcttggcgta
     3001 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     3061 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3121 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3181 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3241 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     3301 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     3361 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     3421 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     3481 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     3541 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     3601 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     3661 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     3721 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     3781 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     3841 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     3901 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     3961 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4021 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4081 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4141 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4201 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4261 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     4321 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     4381 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     4441 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     4501 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     4561 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     4621 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     4681 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     4741 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     4801 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     4861 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     4921 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     4981 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5041 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5101 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     5161 cgtctaagaa accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc
     5221 ctttcgtc
//
