LOCUS       sequence-360420-        6926 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses FLAG tagged FZD4 swapped with FZD3 CRD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6926)
  TITLE     HOLD - Choi lab 2022
REFERENCE   2  (bases 1 to 6926)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pcDNA FZD4_3CRD
FEATURES             Location/Qualifiers
     source          1..6926
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..74
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(193..213)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        198..527
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      378..513
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     440..459
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             594..1388
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(648..667)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1258..1277
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1564..1685
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1601..1620)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1655..1674
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1734..1750)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1734..1750)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1747..1769)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1758..1774
                     /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(join(1782..1788,1789..1806,1807..1812))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1827..1848
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1965..1982)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2136..2724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2216..2235)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2895..3686,3687..3755))
                     /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     3518..3537
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3756..3860)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3935..3954)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4126..4505
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4506..4709
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4660..4680
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4754..4773
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4754..4772
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4806..4850
                     /codon_start=1
                     /product="leader sequence from mouse preprotrypsin"
                     /label=preprotrypsin leader
                     /translation="MSALLILALVGAAVA"
     CDS             4851..4874
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6366..6383
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6406..6423)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6412..6636
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(6682..6926,1..184)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6769..6788)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
       61 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
      121 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
      181 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
      241 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
      301 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
      361 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
      421 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
      481 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
      541 cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt cgcatgattg
      601 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
      661 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
      721 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
      781 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
      841 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
      901 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
      961 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     1021 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     1081 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     1141 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     1201 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     1261 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     1321 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     1381 tcttctgagc gggactctgg ggttcgcgaa atgaccgacc aagcgacgcc caacctgcca
     1441 tcacgagatt tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc
     1501 cgggacgccg gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac
     1561 cccaacttgt ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc
     1621 acaaataaag catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta
     1681 tcttatcatg tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag
     1741 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1801 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1861 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1921 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1981 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     2041 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     2101 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2161 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2221 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2281 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2341 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2401 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2461 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2521 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2581 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2641 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2701 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2761 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2821 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2881 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2941 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3001 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     3061 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3121 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3181 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3241 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3301 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3361 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3421 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3481 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3541 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3601 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3661 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3721 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3781 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3841 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt cgacggatcg
     3901 ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat gccgcatagt
     3961 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     4021 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     4081 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     4141 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     4201 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     4261 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     4321 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     4381 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     4441 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     4501 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     4561 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     4621 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     4681 tacggtggga ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact
     4741 ggcttatcga aattaatacg actcactata gggagaccca agctggctag ttaagcttgg
     4801 taccaatgtc tgcacttctg atcctagctc ttgttggagc tgcagttgct gactacaaag
     4861 acgatgacga caagcttgcg gccgcacaca gtttgttttc ttgtgaacct attaccttga
     4921 ggatgtgcca agatttgcct tataatacta ccttcatgcc taatcttctg aatcattatg
     4981 accaacagac agcagctttg gcaatggagc cattccaccc tatggtgaat ctggattgtt
     5041 ctcgggattt ccggcctttt ctttgtgaac tctacgctcc tatttgtatg gaatatggac
     5101 gtgtcacact tccctgtcgt aggctgtgtc agcgggctta cagtgagtgt tcgaagctca
     5161 tggagatgtt tggtgttcct tggcctgaag atatggaatg cagtaggttc ccagattgtg
     5221 atgagggacc aggtgatgaa gaggttccct tgccccacaa gactcccatc cagcccgggg
     5281 aagagtgcca ctccgtggga agcaattctg atcagtacat ctgggtgaag aggagcctga
     5341 actgtgttct caagtgtggc tacgatgctg gcttgtacag ccgctcagct aaggagttca
     5401 cggatatttg gatggctgtg tgggccagcc tctgcttcat ctccaccacc ttcaccgtgc
     5461 tgaccttcct gattgattca tccaggtttt cttaccctga gcgccccatc atatttctca
     5521 gtatgtgcta taatatttat agcattgctt atattgttcg gctgactgta ggccgggaaa
     5581 ggatatcctg tgattttgaa gaggcggcag agcccgttct catccaagaa ggacttaaga
     5641 acacaggatg tgcaataatt ttcttgctga tgtacttttt tggaatggcc agctccattt
     5701 ggtgggttat tctgacactc acttggtttt tggcagccgg actcaagtgg ggtcatgaag
     5761 ccattgaaat gcacagttct tatttccaca tcgcagcctg ggctattccc gcagtgaaaa
     5821 ccattgtcat cttgattatg agactagtgg atgccgatga actgactggc ttgtgctatg
     5881 ttgggaacca aaacctagat gccctcactg gctttgtggt ggctcctctc tttacgtatt
     5941 tggtgattgg aacgctgttc attgcggcgg gtttggtggc cttattcaaa attcggtcca
     6001 atcttcaaaa agacgggaca aagacagaca agttggaaag gctaatggtc aagatcgggg
     6061 tcttctcagt actgtacacg gttcctgcaa cctgtgtgat tgcctgttat ttctatgaaa
     6121 tctcaaactg ggcactcttt cgatattctg cagatgactc aaacatggca gttgaaatgt
     6181 tgaaaatttt tatgtctttg ctcgtgggca tcacttcagg catgtggatt tggtctgcca
     6241 aaactcttca cacgtggcaa aagtgttcta accgattggt gaattctggg aaggtaaaga
     6301 gagagaagag ggggaatggt tgggtgaagc caggaaaagg caacgagact gtggtatagc
     6361 tcgagcatca tcaccatcac cattgagttt aaacccgctg atcagcctcg actgtgcctt
     6421 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     6481 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     6541 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     6601 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     6661 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     6721 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     6781 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     6841 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     6901 gtgatggttc acgtagtggg ccatcg
//
