LOCUS       Exported                5164 bp ds-DNA     circular SYN 08-APR-2021
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5164)
  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..5164
                     /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     282..302
                     /label=pRP LHA Seq (Fw) - LP7982
     misc_feature    377..401
                     /label=uni-LHA-gib-A
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     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..935
                     /label=TSC22D2-Cterm-no stop codon
     CDS             678..935
                     /codon_start=1
                     /label=exon 4
                     /translation="DLVKSHLMYAVREEVEVLKEQIKELVERNSLLERENALLKSLSSN
                     DQLSQLPTQQANPGSTSQQQAVIAQPPQPTQPPQQPNVSSA"
     misc_feature    complement(909..928)
                     /label=guide 148 - modified
     misc_feature    complement(912..931)
                     /label=guide 220 -modified
     misc_feature    912..917
                     /label=2 silent muts
     misc_feature    complement(931..935)
                     /label=guide 219-a
     misc_feature    936..2375
                     /label=mScarlet-PCR
     CDS             936..962
                     /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"
     misc_feature    936..960
                     /label=uni-LHA-gib-B
     primer_bind     936..956
                     /label=C-Cassette (Fw) - LP7888
     CDS             963..1655
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet-I
                     /note="matures faster than mScarlet"
                     /translation="VSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFSWDILSPQFMYGSRAFIKHPADIPDYYKQSFPEGFKWERVMNFEDGGA
                     VTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIKM
                     ALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRHS
                     TGGMDELYK"
     primer_bind     complement(1093..1111)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1361..1380
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1662..1679
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1680..1688
                     /codon_start=1
                     /label=GSG-2A
                     /note="N-terminal GSG for efficient P2A transition. 
                     Modified to include BamHI site. "
                     /translation="GSG"
     CDS             1689..1745
                     /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     1689..1713
                     /label=P2A (Fw)
     primer_bind     1689..1713
                     /label=P2A (Fw) - LP7890
     primer_bind     complement(1725..1745)
                     /label=P2A (Rev)
     primer_bind     complement(1725..1745)
                     /label=P2A (Rev) - LP7891
     CDS             1752..2123
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=Zeo
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="AKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDDV
                     TLFISAVQDQVVPDNTLAWVLVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGRE
                     FALRDPAGNCVHFVAEEQD"
     polyA_signal    2130..2354
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     misc_feature    2351..2375
                     /label=uni-RHA-a
     misc_feature    2355..2375
                     /label=Universal seq
                     /note="Cterm. To allow for easy and universal switching of 
                     resistances with a primer. Can remove or replace polyA 
                     signal (SV40, etc.). Allow for universal Gibson homology 
                     for RHA, with High-flex linker providing homology for LHA."
     primer_bind     complement(2355..2375)
                     /label=C-Cassette (Rev) - LP7889
     CDS             2376..2924
                     /codon_start=1
                     /label=TSC22D2-RHA
                     /translation="*SFLKPH*EKN*KQSILVCHWCSFHFIRKQVAMLWLCVWPFQY*T
                     IILQELFLSLRCHAALLMSSSMSSVHKENNRWGLLKRAKSAFYLVRMSGVFKSFGGSPM
                     FPITHGFTLSLPITL*ITVHLKSHFSF*FSNICLHKFSFICVLFITGLLFS*CT*TMSQ
                     NFFNFFE*L*VSVKEVKDRI"
     misc_feature    2376..2390
                     /label=guide 219-b
     misc_feature    2925..2949
                     /label=uni-RHA-b
     misc_feature    2926..2930
                     /label=MCS
                     /note="pUC19 multiple cloning site"
     primer_bind     complement(2943..2959)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2943..2959)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    2967..2983
                     /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)."
     promoter        complement(2991..3021)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     primer_bind     complement(3025..3045)
                     /label=pRP RHA Seq (Rev) - LP7983
     rep_origin      complement(3345..3933)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(4104..4964)
                     /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"
     promoter        complement(4965..5069)
                     /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 cttttttttt aattctaagc
      421 ttagttgaca atttaagaag ctagtatttt ttggaaatcg tcccaaactt tgaaaagaaa
      481 attctgggct tactgctctg gttgggaact ggattttttc caacccatct tccataggct
      541 cggtcaagat aaagcagaaa cattaaaagc aagagcagga aaactagtat aaaaacttag
      601 caccaagtag gccagaacca gatagcacca cctttatctt ttcactcttt tgacattcct
      661 aattttgttt ttcacaggat ctggtgaaaa gccatttgat gtatgcagta agagaagaag
      721 tggaagtttt aaaggaacaa ataaaagaat tagttgaaag aaactcttta cttgaacgag
      781 aaaatgcact gttaaaatct ctttcaagca atgatcaatt atcccaactc ccaacccaac
      841 aggccaatcc tggtagcact tctcaacagc aagcagtgat agcacagcct ccgcagccaa
      901 cgcaacctcc acaacaaccg aatgtctcct cagcaggagg aagcggagga tctggcggta
      961 ccgtgagcaa gggcgaggca gtgatcaagg agttcatgcg gttcaaggtg cacatggagg
     1021 gctccatgaa cggccacgag ttcgagatcg agggcgaggg cgagggacga ccctacgagg
     1081 gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc tcctgggaca
     1141 tcctgtcccc tcagttcatg tacggctcca gggccttcat caagcacccc gccgacatcc
     1201 ccgactacta taagcagtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg
     1261 aggacggcgg cgccgtgacc gtgacccagg acacctccct ggaggacggc accctgatct
     1321 acaaggtgaa gctccgcggc accaacttcc ctcctgacgg ccccgtaatg cagaagaaga
     1381 caatgggctg ggaagcgtcc accgagcggt tgtaccccga ggacggcgtg ctgaagggcg
     1441 acattaagat ggccctgcgc ctgaaggacg gcggacgcta cctggcggac ttcaagacca
     1501 cctacaaggc caagaagccc gtgcagatgc ccggcgccta caacgtcgac cgcaagttgg
     1561 acatcacctc ccacaacgag gactacaccg tggtggaaca gtacgaacgc tccgagggcc
     1621 gccactccac cggaggcatg gacgagctgt ataagctgca gcaccatcac caccatcacg
     1681 gatccggagc tactaacttc agcctgctga agcaggccgg agacgtggag gagaaccctg
     1741 gaccttctag agccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg
     1801 gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact
     1861 tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg
     1921 tgccggacaa caccctggcc tgggtgctgg tgcgcggcct ggacgagctg tacgccgagt
     1981 ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg
     2041 gcgagcagcc gtgggggagg gagttcgccc tgcgcgatcc ggccggcaac tgcgtgcact
     2101 tcgtggccga ggagcaggac taactcgagc tgtgccttct agttgccagc catctgttgt
     2161 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     2221 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     2281 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     2341 ggtgggctct atgggcggcc gctaagagca agaagtaaag ctttcttaag cctcattaag
     2401 aaaaaaactg aaagcaatct atccttgtgt gccactggtg ttctttccac tttatacgaa
     2461 agcaagtagc catgctttgg ttgtgtgttt ggccttttca gtattagaca atcattctac
     2521 aagagctttt cctctctctg agatgtcatg cagcgctgtt gatgtccagt tctatgtcat
     2581 cagtacacaa ggagaataat agatggggtt tattaaagcg agcaaagtct gcattttacc
     2641 tggtgcgcat gagtggggtc tttaagagtt ttggtggctc tcccatgttt cctattaccc
     2701 atggatttac cctgagcctt cctatcacat tataaataac agttcatcta aagagccact
     2761 tttctttctg attcagtaac atttgcctac ataagttttc atttatttgt gttttattta
     2821 ttacagggct gctattttca taatgtacat gaacaatgtc acagaacttt tttaattttt
     2881 ttgaataatt ataagtatca gtaaaggaag tgaaagacag gattaagctt ggcgtaatca
     2941 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     3001 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     3061 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     3121 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     3181 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     3241 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     3301 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     3361 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     3421 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     3481 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     3541 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     3601 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     3661 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     3721 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3781 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3841 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3901 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3961 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     4021 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     4081 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     4141 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     4201 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     4261 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     4321 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     4381 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     4441 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     4501 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     4561 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     4621 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     4681 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     4741 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4801 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4861 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4921 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4981 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     5041 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     5101 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     5161 cgtc
//
