LOCUS       sequence-444973-        5668 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Perform knock-in by homologous recombination in giantin/GOLGB1 
            gene..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5668)
  AUTHORS   Fourriere L, Divoux S, Roceri M, Perez F, Boncompain G
  TITLE     Microtubule-independent secretion requires functional maturation of 
            Golgi elements.
  JOURNAL   J Cell Sci. 2016 Sep 1;129(17):3238-50. doi: 10.1242/jcs.188870. 
            Epub 2016 Jul 13.
  PUBMED    27411366
REFERENCE   2  (bases 1 to 5668)
  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"; doi: 
            "10.1242/jcs.188870"; journalName: "J Cell Sci"; date: "2016-09-1- 
            1"; volume: "129"; issue: "17"; pages: "3238-50"
COMMENT     Sequence Label: GFP-giantin-ATG_donor
FEATURES             Location/Qualifiers
     source          1..5668
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      380..968
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     869..888
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1122..1139
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    1256..1277
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(1292..1297,1298..1315,1316..1322)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1330..1346
                     /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     1335..1357
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     1354..1370
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1354..1370
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1389..1409
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1391..1409
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     1438..1454
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1439..1455
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(2481..2483,2484..3194)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2481..2483,2484..3194)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2523..2544)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2784..2803)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3131..3152
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4196..4212)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4196..4212)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(4244..4263)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4245..4263)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4273..4290)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4273..4289)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4282..4304)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4431..4886
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4518..4537)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4728..4749
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4912..5016
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5017..5085,5086..5668,1..209)
                     /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     complement(5235..5254)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc
       61 gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag
      121 ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga
      181 taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt
      241 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
      301 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
      361 aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa
      421 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt
      481 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc
      541 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa
      601 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa
      661 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc
      721 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa
      781 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa
      841 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg
      901 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc
      961 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg
     1021 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg
     1081 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg
     1141 aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat
     1201 gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg
     1261 tgagttagct cactcattag gcaccccagg ctttacactt tatgctcccg gctcgtatgt
     1321 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
     1381 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggtaccggg ccccccctcg
     1441 aggtcgacgg tatcgataag cttgatatcg aattccagta atttgtgtct tctctgtttt
     1501 tttcttggtc atcttgacta gagttttatc aattttattg atcttttcag ataatcaact
     1561 tttggtttta ttgatattct ctatttttct atttttatta atttctactc tgatttttat
     1621 ttttattttt ctgcttacac tggctgtaat ttgttcttct ttttctaatt ttctatagaa
     1681 gaatcatagg ttactgattt ctgtccttcc tccttttcta atatatgcat ttaatgctgt
     1741 acattttgaa caaagtttat atttaaattt tagcaaagcc acatggttta tcaagaaagc
     1801 tttctgaaag aatggggttc atggggcaca taaaaagaaa aatgttgcag tatcatggag
     1861 tttatttgaa caagtgggag agaataacag tctgacagag gaagaaagaa taagacattc
     1921 atgtttgggc atgtgcctgt agtctcagct actcgaaagg ctgaggtggc aggatcgctt
     1981 gagcccagaa agcagaggtt gcagtaggct gagattgcgt ccctgtactc cagcctgcgt
     2041 gacagagcaa gaccctgtct caaaaaaaaa aaaagaaaaa gaaaaaagaa attcatgttt
     2101 gtaagtgaaa ctactatgaa agaagggcat taaagataaa aactaccttt aaggtgtatt
     2161 taacaattat agattggtat ttaacattgt attaatgttt aacgattagg tacttacatt
     2221 ttattttaga aatggtgtta gtgatggtga taacctctgt acaattgcca ctgaaaagaa
     2281 cagcgtgtga ctacactgga agtactcaaa tgcctttttg aagacatcat tgtgctgcag
     2341 actgtgtcta atacaaatca cctgtacagt attctttttt cattcacgga tgatatggtt
     2401 atcaatatag gagaagccta tgttcatttt gctgattctt aggttttttc cccccttctt
     2461 ttcctacaga aatgctgagc gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     2521 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     2581 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     2641 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     2701 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     2761 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     2821 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     2881 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     2941 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     3001 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     3061 tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga
     3121 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     3181 acgagctgta caagcgatta tcaggattag caaatgttgt tttgcatgaa ttatcaggag
     3241 atgatgacac tgatcagaat atgagggctc ccctagaccc tgtgagtagt tctgttctga
     3301 tttaaactgg taagacatat gttctgtgtt cttctaagga aaataattta agatagaagg
     3361 gtgtgcgaag cctccctggt actaggtgaa gcgaggatta agaaaaatat tgaaatttta
     3421 taagtgatcc ttctcttgta agtttttagc ttaaaagcca cagtgatggg aaacaagtcc
     3481 tggatacaat ttgaaagggc tttctcattt gtctccctgg gccttgtttg gtgccagagt
     3541 gttttaagtc ctttgttctc atccttggct attccttttc cacaggaaag gtctaagtga
     3601 caaatgagga aactggaaca ccttttgggt ttcagaatat ctgtttgctt tgtaatttca
     3661 tttaccactt ctcctatagt ttgtattgta ttttcttttt tttttaagat gatacaaatt
     3721 ggcagtatgt attatatttt ctgaagtcca cataaggaat taataaaact tcttagtttt
     3781 ctgaaaagac aagaagtaat gcgtagtggt taagagagtt ttacttaact actcttaatt
     3841 ttaacttttg gccttttacc cttactggtt tatgattttt gctttccttt atttttctct
     3901 ttggtaaaat taggataata ataccttaaa tgggataatg tttgtaaaat gcttaacatt
     3961 tataaaatac ttaatatatt gtaatggcta tgataatgat tattattgga tcagagttag
     4021 attcttaagt taacaagaat atgaattggg caaaacataa aaagttggaa tgaagaaact
     4081 cctgttggga agctaatacc ttttcctggt gcactggcaa cttttttcct aaatattagg
     4141 aattacacca agaatctgac atggaattta ataatactac acaagaagat gttcggatcc
     4201 actagttcta gagcggccgc caccgcggtg gagctccaat tcgccctata gtgagtcgta
     4261 ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac
     4321 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc
     4381 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg
     4441 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     4501 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     4561 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     4621 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     4681 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     4741 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     4801 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     4861 taacaaaata ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc
     4921 cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc
     4981 tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc
     5041 gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg
     5101 gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat
     5161 ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc
     5221 acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa
     5281 ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa
     5341 aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt
     5401 gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct
     5461 tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat
     5521 gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg
     5581 cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg
     5641 atggaggcgg ataaagttgc aggaccac
//
